Novel treatment for pathologies associated with oxidative damage

ABSTRACT

This invention relates to compositions and methods for the treatment of pathologies associated with intracellular polyamine dysregulation. In particular, the present invention provides compositions and methods involving mammalian polyamine oxidase (PAO) to treat cancer, cell damage, tissue damage caused by ischemia/reperfusion, inflammation, traumatic brain injury, stroke and tissue developmental disorders. Methods for diagnosis and prognosis of cancer and other diseases are also provided by the present invention.

This invention was made, in part, with Government support by theDepartment of Veterans Affairs and the Heart, Lung, and Blood Instituteof the National Institutes of Health, Grant No. HL16251. Accordingly,the Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for the treatment ofpathologies associated with intracellular polyamine dysregulation. Inparticular, the present invention provides compositions and methodsinvolving mammalian polyamine oxidase (PAO) to treat cancer, celldamage, tissue damage caused by ischemia and reperfusion, inflammation,traumatic brain injury, stroke, and tissue developmental disorders.Methods for diagnosis and prognosis of cancer and other diseases arealso provided by the present invention.

BACKGROUND OF THE INVENTION

Diamine, putrescine (Put) and the polyamines, spermine (Spm) andspermidine (Spd) have been implicated in numerous fundamentallyimportant cellular processes, including wound healing, tissuedifferentiation, and tumor growth (Seiler, Prog. Brain Res. 106:333-344[1995]; Wallace, Biochem. Soc. Trans. 26:569-571 [1998]; and Morgan,Biochem. Soc. Trans. 26:586-571 [1998]). A high level of these amines isassociated with cell growth, while a decrease in their levels isassociated with growth inhibition and cell death. The intracellularpolyamine level is strictly regulated by the modulation of enzymesinvolved in their biosynthesis, catabolism and transport (Pegg, Biochem.J. 234:249-262 [1986]; and Casero and Pegg, FASEB J. 7:652-661 [1993]).

Lowered levels of Put, Spm, and Spd, result from the inhibition ofornithine decarboxylase (ODC) or polyamine oxidase (PAO) (Hölttä,Biochemistry 16:91-100 [1977]; Bolkenius and Seiler, Int. J. Biochem.13:287-292 [1981]; Hölttä, Methods Enzymol. 94:306-311 [1983]; Seiler,supra [1995]), or from high acetyl Coenzyme A:spermidine/spermine-N¹-acetyltransferase (SSAT) activity. In cells ofhigher organisms, ODC converts ornithine to Put, which is then convertedto Spd by Spd synthase. Spd in turn is converted to Spm by Spm synthase.SSAT acetylates Spd and Spm for excretion from cells or for oxidation bythe peroxisomal flavoprotein PAO (van den Munckhof et al., J. Histochem.Cytochem. 43:1155-1162 [1995]). PAO oxidizes N¹-acetyl-Spm to3-acetamidopropanal and Spd, and N¹-acetyl-Spd to 3-acetamidopropanaland Put. Spd and Put return to the intracellular polyamine pool, and3-acetamidopropanal can be deacetylated to form the cytotoxin,3-aminopropanal (Houen et al., Acta Chem. Scand. 48:52-60 [1994]).Importantly, 3-aminopropanal is thought to contribute, either alone orin concert with H₂O₂, to tissue damage following traumatic or ischemicinjury (Ivanova et al., J. Exp. Med. 188:327-340 [1998]; Dogan et al.,J. Neurosurg. 90:1078-1082 [1999]; and Dogan et al., J. Neurochem.72:765-770 [1999]; Ivanova et al., Proc. Natl. Acad. Sci. USA99:5579-5584 [2002]). On exposure to substrate, the flavin adeninedinucleotide (FAD) of PAO becomes reduced. As shown in FIG. 1, thereduced FAD is reoxidized by O₂ to regenerate active PAO and to producehydrogen peroxide. Through production of hydrogen peroxide, PAOregeneration has been proposed to play a role in triggering and/orparticipating in the progression of apoptosis (Hu and Pegg, Biochem J.328:307-316 [1997]; Kramer et al., Cancer Res. 59:1278-1286 [1999];Mank-Seymour et al., Clin. Cancer Res. 4:2003-2008 [1998]; Lindsay andWallace, Biochem. J. 337:83-87 [1999]; and Chopra and Wallace, BiochemPharmacol. 55:1119-1123 [1998]).

Thus, mammalian PAO has clinical and pharmacological relevance tovarious pathological conditions. Clearly, there is a need in the art forthe development of molecular and biochemical tools to produce PAO-basedcompositions and methods for diagnostic, prognostic and therapeuticapplications.

SUMMARY OF THE INVENTION

This invention relates to compositions and methods for the treatment ofpathologies associated with intracellular polyamine dysregulation. Inparticular, the present invention provides compositions and methodsinvolving mammalian polyamine oxidase (PAO) to treat cancer, celldamage, tissue damage caused by ischemia and reperfusion, inflammation,traumatic brain injury, stroke, and tissue developmental disorders.Methods for diagnosis and prognosis of cancer and other diseases arealso provided by the present invention.

The present invention provides isolated nucleic acids that comprise anopen reading frame for a peroxisomal polyamine oxidase of a mammal. Inpreferred embodiments, the mammal is selected from the group consistingof cattle, mice and humans. In particularly preferred embodiments, theisolated nucleic acid is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5. In some embodiments the isolatednucleic acid comprises deoxyribonucleic acid.

In addition, the present invention provides vectors comprising at leastone isolated nucleic acid selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5. In some embodiments, the vectorfurther comprises a promoter and an operator operatively linked to thenucleic acid. In some preferred embodiments the vector is a bacterialexpression vector.

The present invention also provides compositions comprising at least onerecombinant peroxisomal polyamine oxidase of a mammal. In preferredembodiments, the mammal is selected from the group consisting of cattle,mice and humans. In some particularly preferred embodiments, therecombinant peroxisomal polyamine oxidase comprises a sequence selectedfrom the group consisting of SEQ ID NO:4 and SEQ ID NO:6. In otherembodiments, the recombinant peroxisomal polyamine oxidase furthercomprises a polyhistidine tag.

In some embodiments, the present invention provides host cellstransformed with a vector comprising a nucleic acid selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. In somepreferred embodiments, the host cell is a bacterial cell. In someparticularly preferred embodiments, the bacterial cell is an E. colicell.

The present invention also provides compositions comprising thepolyamine oxidase produced by a host cell transformed with a vectorcomprising a nucleic acid selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5. In some embodiments, the compositionfurther comprises ethylene glycol. In some preferred embodiments, thepolyamine oxidase of the composition further possesses enzymaticactivity.

In addition, the present invention provides methods for detectingpolyamine oxidase expression in a cell comprising the steps of: a)providing: at least one cDNA generated from mRNA harvested from thecell, at least one polyamine oxidase primer pair, and at least onecontrol primer pair; b) annealing the cDNA with the polyamine oxidaseprimer pair and amplifying the cDNA under conditions such that anamplified polyamine oxidase DNA fragment is obtained; and c) annealingthe cDNA with the control primer pair such that an amplified control DNAfragment is obtained. In some embodiments, the amplification isconducted by polymerase chain reaction. In some embodiments, the methodfurther comprises electrophoresis of the amplified DNA fragments throughan agarose gel. In related embodiments, the method further comprisesstaining the amplified DNA fragments within the gel with ethidiumbromide and measuring the fluorescence intensity of the ethidiumbromide-stained DNA fragments. In some preferred embodiments, themethods further comprise step d) comparing the fluorescence intensity ofthe ethidium bromide-stained polyamine oxide DNA fragment to theethidium bromide-stained control DNA fragment.

Moreover, the present invention provides isolated nucleic acids thatcomprises a sequence selected from the group consisting of a geneencoding a peroxisomal acetylpolyamine oxidase protein of a mammal, agene encoding a biologically active portion of the peroxisomalacetylpolyamine oxidase protein, and a gene encoding a biologicallyactive variant of the peroxisomal acetylpolyamine oxidase protein. Insome embodiments, the mammal is selected from the group consisting of acow, a mouse and a human. In related embodiments, the nucleic acid isselected from the group consisting of the open reading frames of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5. In further embodiments, the nucleicacid encodes a protein selected from the group consisting SEQ ID NO:2,SEQ ID NO:4, and SEQ ED NO:6. In some preferred embodiments, the nucleicacid encodes a protein with amine oxidizing activity. In a subset ofthese embodiments, the substrate for the amine oxidizing activity isselected from the group consisting of N¹-acetyl-Spm, N¹-acetyl-Spd,N¹,N¹²-diethyl-Spm, N¹,N¹¹-diethyl-nor-Spm, and Spm. Also provided arevectors comprising an isolated nucleic acid sequence selected from thegroup consisting of a gene encoding a peroxisomal acetylpolyamineoxidase protein of a mammal, a gene encoding a biologically activeportion thereof, and a gene encoding a biologically active variantthereof. In some embodiments, the vector further comprises a promoteroperatively linked to the nucleic acid. Additionally, the presentinvention provides host cells transformed with a vector comprising anisolated nucleic acid sequence selected from the group consisting of agene encoding a peroxisomal acetylpolyamine oxidase protein of a mammal,a gene encoding a biologically active portion thereof, and a geneencoding a biologically active variant thereof. In some embodiments, thehost cell is located in an animal. In other embodiments, the presentinvention provides a host cell comprising a disruption of a geneencoding a peroxisomal acetylpolyamine oxidase protein of a mammal, agene encoding a biologically active portion thereof, and a gene encodinga biologically active variant thereof. In some embodiments, the hostcell is located in an animal.

In some embodiments, the present invention provides isolated mammaliannucleic acid sequences selected from the group consisting of the openreading frames of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and sequencesthat hybridize to the complement of the open reading frames of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, under conditions of low stringency, andwherein the isolated nucleic acid sequence encodes a polypeptide havingamine oxidizing activity.

In other embodiments, the present invention provides compositionscomprising an isolated protein selected from the group consisting of aperoxisomal acetylpolyamine oxidase of a mammal, a biologically activeportion thereof, or a biologically active variant thereof. In somepreferred embodiments, the mammal is selected from the group consistingof a cow, a mouse and a human. In a subset of these embodiments, theperoxisomal acetylpolyamine oxidase comprises a sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Insome preferred embodiments, the peroxisomal acetylpolyamine oxidase hasamine oxidizing activity. In some of these embodiments, the substratefor the amine oxidizing activity is selected from the group consistingof N¹-acetyl-Spm, N¹-acetyl-Spd, N¹,N¹²-diethyl-Spm, N¹,N¹¹-diethyl-nor-Spm, and Spm. Moreover, the present invention providesembodiments wherein the peroxisomal acetylpolyamine oxidase is arecombinant peroxisomal acetylpolyamine oxidase protein. In relatedembodiments, the peroxisomal acetylpolyamine oxidase protein furthercomprises an affinity tag.

The present invention provides methods for detecting mammalianperoxisomal acetylpolyamine oxidase expression in a cell comprising thesteps of: providing: i) a sample from a mammalian subject, and ii) atleast one reagent capable of specifically detecting mammalianperoxisomal acetylpolyamine oxidase expression; and contacting thesample with at least one reagent under conditions suitable for bindingat least one reagent to a mammalian peroxisomal acetylpolyamine oxidasegene product. In embodiments in which the mammalian peroxisomalacetylpolyamine oxidase gene product comprises mRNA, at least onereagent comprises a nucleic acid probe of at least 12 nucleotides inlength that specifically hybridizes under conditions of high stringencyto the mRNA or to cDNA corresponding to the mRNA. In relatedembodiments, the contacting is accomplished by a technique selected fromthe group consisting of polymerase chain reaction and Northern blotting.In embodiments in which the mammalian peroxisomal acetylpolyamineoxidase gene product comprises protein, the at least one reagentcomprises an antibody that binds to the protein. In related embodiments,the contacting is accomplished by a technique selected from the groupconsisting of enzyme-linked immunosorbent assay, Western blotting,immunofluorescence analysis, immunohistochemistry and flow cytometry. Ina subset of these embodiments, the antibody further comprises a reportermolecule selected from the group consisting of an enzyme and afluorochrome.

Moreover the present invention provides methods of inhibiting mammalianperoxisomal acetylpolyamine oxidase activity comprising: providing amammalian peroxisomal acetylpolyamine oxidase, and an inhibitor; andcontacting the mammalian peroxisomal acetylpolyamine oxidase with theinhibitor under conditions suitable for reducing amine oxidizingactivity of the oxidase. In some preferred embodiments, the inhibitor isselected from the group consisting of synthalin andN-(3-aminopropyl)-1,10 decanediamine. In further embodiments, themammalian peroxisomal acetulpolyamine oxidase is located in a cell or inan animal.

Additionally, the present invention provides methods comprising:providing a host cell comprising an exogenous nucleic sequence selectedfrom the group consisting of the open reading frames of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5 and sequences that hybridize to the complement ofthe open reading frames of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 underconditions of low stringency, wherein the isolated nucleic acid sequenceencodes a polypeptide having amine oxidizing activity; and culturing thehost cell under conditions such that the exogenous nucleic acid sequenceis expressed.

DESCRIPTION OF THE FIGURES

The following Figures form part of the Specification and are included tofurther demonstrate certain aspects and embodiments of the presentinvention. The invention may be better understood by reference to one ormore of these Figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 depicts substrate oxidation by PAO. Panel A shows N¹-acetyl-Spmoxidation by PAO, while panel B shows N¹-acetyl-Spd oxidation by PAO.FADH₂ is the two-electron reduced form of the enzyme bound FAD, while Rstands for the ribityl-diphosphoadenine moiety of FAD. Also shown is thein vivo deacetylation of 3-acetamidopropanal to the cytotoxin,3-aminopropanal, and the conversion of H₂O₂ to H₂O and O₂ by catalase.

FIG. 2 shows the partial bovine PAO (bPAO) cDNA (SEQ ID NO:1) and aminoacid sequences (SEQ ID NO:2). The dashes indicate a segment of theprotein sequence of unknown length and composition, the asterisk denotesthe translation stop codon, and the double underline denotes theperoxisomal transport signal. The three single-underlined amino acidsegments were derived by protein sequencing of the intact enzyme (SEQ IDNO:7) and two tryptic peptides (SEQ ID NO:8; and SEQ ID NO:9).

FIG. 3 shows the murine PAO (mPAO) cDNA (SEQ ID NO:3) and amino acidsequences (SEQ ID NO:4). The + symbol marks the start of exon I. Thisbase (No. 18) corresponds to base No. 7477 of SEQ ID NO:21 and base No.1069500 of GenBank Accession No. NW₁₃ 000335 (gene transcript ID No. XM133921.1). The double-underline at the 5′-end represents sequence fromthe cloning vector, the asterisk denotes the translation stop codon, andthe single-underline denotes the peroxisomal transport signal.

FIG. 4 shows the human (hPAO) PAO cDNA (SEQ ID NO:5) and amino acidsequences (SEQ ID NO:6). The + symbol represents the start of the exonI. This base (No. 27) corresponds to base No. 2840 of SEQ ID NO:14 andbase No. 83941 of GenBank Accession No. AL360181.31. Thedouble-underline at the 5′-end represents sequence from the cloningvector, the asterisk denotes the translation stop codon, and thesingle-underline denotes the peroxisomal transport signal.

FIG. 5 shows an alignment of the human (complete), murine (complete) andbovine (partial) PAO amino acid sequences. The underlined segments ofthe bovine sequence indicate the sequences derived from Edmandegradation analysis of the intact protein (amino-terminus) (SEQ IDNO:8) and two pure peptides (SEQ ID NO:9 and SEQ ID NO:10). Thedouble-underlined bPAO segment indicates the overlap of information frompeptide sequencing and from translation of the cDNA sequence. At theC-termini, the Pro-Arg-Leu motif is a peroxisomal transport signal. Thequestion marks for the bovine sequence indicate a segment of unknowncomposition.

FIG. 6 provides schematics of human and murine PAO mRNA and genomic DNA.Panel A depicts a schematic of human PAO mRNA. Panel B depicts aschematic of the human PAO genomic DNA, which is located on chromosome10 and contains 7 exons totaling 1822 bp, including an openreading-frame of 1533 bp. Introns are represented by lines, while exonsare represented by boxes with the length of each exon in base pairslisted above. Panel C provides a schematic of the human PAO exons withnumbers indicating the locations of the putative splice sites. Thenumber at the beginning of exon I corresponds to base No. 27 of the hpaocDNA sequence in FIG. 4, and base No. 2840 of the hpao genomic sequenceof SEQ ID NO:14. The number at the end of exon VII corresponds to baseNo. 1849 of the cDNA sequence in FIG. 4 and base No. 15266 of thegenomic sequence of SEQ ID NO:14. Panel D depicts the location of adeletion in an EST derived from a genitourinary high-grade transitionalcell tumor (GenBank Accession No. AW662266), the wild type and mutanthuman sequences are also disclosed herein as SEQ ID NO:22 and SEQ IDNO:23, respectively. Panel E depicts a schematic of murine PAO mRNA.Panel F shows a schematic of murine PAO genomic DNA (length of 8651 bp),which is located on chromosome 7 and contains 7 exons totaling 1755 bp,including an open reading-frame of 1512 bp. Introns are represented bylines, while exons are represented by boxes with the length of each exonin base pairs listed above. Panel G provides a schematic of murine PAOexons with numbers indicating the locations of putative splice sites.The number at the beginning of exon I correspond to base No. 18 and ofthe mpao cDNA sequence in FIG. 3, and base No. 7477 of the mpao genomicsequence of SEQ ID NO:21. The number at the end of exon VII correspondsto base No. 1758 of the cDNA sequence in FIG. 3 and base No. 16129 ofthe genomic sequence of SEQ ID NO:21.

FIG. 7, panel A displays the chromosomal location of the human polyamineoxidase gene as well as a cytogenetic map. FIG. 7, panel B shows thechromosomal location of murine polyamine oxidase gene and a cytogeneticmap.

FIG. 8 shows the UV-visible spectrum, recorded at pH 7.6, of recombinantmPAO produced in bacteria.

FIG. 9 show the results of a reductive dithionite titration (DT) ofmPAO. Panel A shows the spectrum of oxidized mPAO (------), includingthose obtained at the beginning of the titration after addition of 2.16and 4.433 nmol DT (solid lines), and that of fully reduced mPAO afteraddition of 17.3 nmol DT ( - - - ). The arrows indicate the change inabsorbance that occurred upon DT addition. Panel B displays the spectralchanges that occurred in the latter portion of the titration. The arrowsindicate the changes that took place as progressively more DT was added:4.33, 6.49, 8.66, 10.8, 13.0, 15.1 and 17.3 nmol DT. The inset shows aplot of absorbance versus DT concentration. Panel C displays the spectraof the fully oxidized (dashed line, - - - ) , the radical (straightline,

), and the fully reduced (dotted line, ------ ) forms of FAD bound tomPAO, which resulted from the Factor Analysis of the titration datapresented in Panels A and B.

FIG. 10 displays structures of substrates and inhibitors of PAO. Theasterisks indicate those compounds that are substrates for mPAO.

FIG. 11 shows an agarose gel containing the mPAO (lower frame) andβ-actin (upper frame) PCR products amplified from murine cDNA fromvarious tissues and developmental stages. The M denotes the lanecontaining the DNA standards, and the numbered lanes represent mRNA fromthe following tissues: 1) brain; 2) heart; 3) kidney; 4) spleen; 5)thymus; 6) liver; 7) stomach; 8) small intestine; 9) muscle; 10) lung;11) testis; 12) skin; 13) adrenal gland; 14) ovary; 15) uterus; 16)prostate gland; 17) 8.5 day old embryo; 18) 9.5 day old embryo; 19) 12.5day old embryo; 20) 19 day old embryo; 21) virgin breast; 22) pregnantbreast; 23) lactating breast; and 24) involuting breast. Embryo ages aregiven in days post-conception.

FIG. 12 shows a multiple tissue Northern blot screened with HPAO (upperpanel) and β-actin (lower panel) hybridization probes.

FIG. 13 provides a comparison of hpao mRNA expression levels in normalhuman liver and placenta, and OVCAR-3 and HL-60 cancer cell lines (toppanel). GAPDH expression was examined in these same mRNA preparations asa positive control (bottom control).

FIG. 14 shows the toxic effect of N¹-acetyl-Spm on OVCAR-3 and HL-60cells. The y-axis represents the percentage of surviving cells, whilethe x-axis represents the final concentration of N¹-acetyl-Spm added tothe culture medium.

FIG. 15 shows the predicted secondary structure of mPAO obtained usingthe “Predict Protein” (PED-sec) and “Psi-Pred” version 2 (Psi-Pred)programs. Also shown is a comparison of these structures with the“refined” secondary structure of the (PDB) three-dimensional model ofmPAO determined as described in Example 5. H indicates alpha helixpropensity, while E indicates beta-sheet propensity.

FIG. 16 shows a stereoview of the theoretical ribbon structures for amPAO/MDL 72527 complex (top panel). The arrows point to theRossmann-fold motif that interacts primarily with the ribityl-ADPportion of FAD. The MDL 72527 inhibitor (bottom panel) is given apositive charge on N10 because there is an apparent strong electrostaticinteraction between this ammonium ion and the carboxylate of Glu⁸⁴. TheC4-carbon corresponds to the center oxidized in the normal substratereaction. One of the C4 hydrogens of the inhibitor is closest to theN5-position of FAD (2.59 Å). MDL 72527 is shown in white, FAD is shownin black, the α-helix (residues 475 to 494) that interacts with theN1/C2O locus is shown in dark gray, and the α-helix (residues 14 to 26)that interacts with the diphosphoryl portion of FAD is shown in white.

FIG. 17 shows the stereoviews of theoretical ribbon structures for amPAO/N¹-acetyl-Spm complex. The top frame shows the modeled structurefrom the same perspective as that of FIG. 16, while the middle frameshows the same structure viewed down the substrate binding pocket, alongthe axis of the stretched out substrate. The top part of the structurein the top frame is the flavin-binding domain, while the bottom part isthe substrate-binding domain. The substrate N16-nitrogen is directlyinside the opening to the binding pocket, while a hydrogen on C6 of thesubstrate is closest to the flavin N5-position (2.70 Å). There arepositive charges on N12 and N16 of the N¹-acetyl-Spm substrate shown inthe bottom frame, because there appears to be specific interactionbetween these amino groups and Glu⁸⁴ and Asp³³⁹. In the stereoviews, thesubstrate is shown in white, and FAD is shown in black.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined and discussed below.

The term “peroxisome” refers to a small organelle found in the cytoplasmof the cell which houses reactions in which toxic peroxides are formedas unavoidable side products of chemical reactions.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends, such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

In particular, the terms “PAO gene” “polyamine oxidase gene”“peroxisomal polyamine oxidase gene” “acetylpolyamine oxidase gene”“N¹-acetylpolyamine oxidase gene” “N¹-acetyl-spermine/spermidine oxidasegene” and “APAO gene” as used herein, refer to full-length mammalianperoxisomal acetylpolyamine oxidase genes. In some preferredembodiments, the term “PAO gene” refers to the nucleotide sequencesdisclosed herein as SEQ ID NO:1 (bPAO), SEQ ID NO:3 (mPAO) alsodisclosed as GenBank Accession No. NM_(—)153783, SEQ ID NO:5 (hPAO), andSEQ ID NO:14 (hPAO). However, it is also intended that the termencompasses fragments of the PAO nucleotide sequence, as well as otherdomains (e.g., functional domains) within the full-length PAO nucleotidesequence. Furthermore, the terms “PAO gene,” “PAO nucleotide sequence,”and “PAO polynucleotide sequence” encompass DNA, cDNA, and RNAsequences. However, in the context of the present invention, the terms“PAO gene” and “polyamine oxidase gene” do not refer to the mammaliancytosolic polyamine oxidase genes (e.g. GenBank Accession Nos.:NM_(—)019025, AL121675, AY033889,AY033890, AY033891, AF519179, AK000753and BC004831 disclosed by Wang et al., Cancer Research, 61:5370-5373[2001]; Murray-Stewart et al., Biochem. J., 368:673-677 [2002]; Vujcic,et al., Biochem. J. 367:665-675 [2002]; and in International PatentApplication No. WO 02/100884, herein incorporated by reference). Thiscytosolic enzyme will be referred to herein as spermine oxidase since itoxidizes Spm but not N¹-acetyl-Spm or N¹-acetyl-Spd, in contrast toAPAO.

As used herein, the terms “PAO protein” “polyamine oxidase protein”“peroxisomal polyamine oxidase protein” “acetylpolyamine oxidaseprotein” “N¹-acetylpolyamine oxidase protein”“N¹-acetyl-spermine/spermidine oxidase protein” and “APAO protein” referto mammalian peroxisomal acetylpolyamine oxidase proteins, includingwild type and mutant PAO proteins, but does not refer to the mammalianSpm oxidase just mention (NCBI Accession Nos. BAA91360, BAA91360,AAK55763 and AAH04831 disclosed by Vujcic, et al. [2002]). The partialbovine PAO protein sequence is set forth as SEQ ID NO:2, the murine PAOprotein sequence is set forth as SEQ ID NO:4, and the human PAO proteinsequence is set forth as SEQ ID NO:6. Some embodiments of the presentinvention comprise mammalian homologs of the human PAO protein, whichdiffer from the human PAO protein in fewer than 25% of the residues(e.g., percent sequence similarity). Other embodiments comprise variantsof the mammalian PAO proteins, which differ from the wild type PAOsequences in fewer than 1% of the residues (e.g., percent sequencesimilarity).

As used herein, the terms “open reading frame,” “ORF,” “codingsequence,” and “coding region” refer to the nucleotide sequences thatencode the amino acid sequences found in the nascent polypeptide as aresult of translation of an mRNA molecule. The coding region is boundedin eukaryotes, on the 5′ side by the nucleotide triplet “ATG” thatencodes the initiator methionine and on the 3′ side by one of the threetriplets which specify stop codons (i.e., TAA, TAG, and TGA).

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the term “purified” refers to molecules (polynucleotidesor polypeptides) that are removed from their natural environment,isolated or separated. “Substantially purified” molecules are at least50% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated.

In particular, the term “isolated” when used in relation to a nucleicacid, as in “an isolated oligonucleotide” or “isolated polynucleotide”refers to a nucleic acid sequence that is identified and separated fromat least one contaminant nucleic acid with which it is ordinarilyassociated in its natural source. Isolated nucleic acid is present in aform or setting that is different from that in which it is found innature. In contrast, non-isolated nucleic acids are nucleic acids suchas DNA and RNA found in the state they exist in nature. The isolatednucleic acid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

Where amino acid sequence is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. The term“wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene.

In contrast, the terms “modified,” “mutant,” and “variant” refer to agene or gene product that displays modifications in sequence and orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the terms “complementary” and “complementarity” refer topolynucleotides related by base-pairing rules. For example, for thesequence “5′-AGT-3′,” the complementary sequence is “3′-TCA-5′.”

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from 12nucleotides to the entire nucleotide sequence minus one nucleotide. Insome embodiments, the term portion refers to nucleic acid fragments ofat least 24 nucleotides in length. In preferred embodiments, thefragments are at least 48 nucleotides in length, in particularlypreferred embodiments, the fragments are at least 96 nucleotides inlength.

The term “portion” as used herein when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid. In someembodiments, the term portion refers to polypeptides of at least 8 aminoacids in length. In preferred embodiments, the polypeptides are at least16 amino acids in length, in particularly preferred embodiments, thepolypeptides are at least 32 nucleotides in length.

As used herein, the term “biologically active” refers to a moleculehaving structural, regulatory and or biochemical functions of a wildtype PAO molecule (e.g., peroxisomal acetylpolyamine oxidase gene orprotein). In some instances, the biologically active molecule is amammalian PAO molecule (e.g., HPAO or its homologs), while in otherinstance the biologically active molecule is a portion of a mammalianPAO molecule. Other biologically active molecules which find use in thecompositions and methods of the present invention include but are notlimited to mutant (e.g., variants with at least one deletion, insertionor substitution) mammalian PAO molecules. Biological activity isdetermined for example, by restoration or introduction of PAO activityin cells which lack PAO activity, through transfection of the cells witha PAO expression vector containing a PAO gene, derivative thereof, orportion thereof. In preferred embodiments, biologically activity isdetermined by measuring amine-oxidizing activity of the PAO variant ofinterest using the methods disclosed in Example 5.

The term “conservative substitution” as used herein refers to a changethat takes place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartame, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, 4th ed., WH Freeman and Co., New York, pp.18-23 [1981]).Whether a change in the amino acid sequence of a peptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner. In contrast, the term “nonconservative substitution”refers to a change in which an amino acid from one family is replacedwith an amino acid from another family (e.g., replacement of a glycinewith a tryptophan). Guidance in determining which amino acid residuescan be substituted, inserted, or deleted without abolishing biologicalactivity can be found using computer programs (e.g., LASERGENE software,DNASTAR Inc., Madison, Wis.).

The term “mammals” refers to animals of the class mammalia which nourishtheir young by fluid secreted from mammary glands of the mother,including human beings. The class “mammalian” includes placentalanimals, marsupial animals, and monotrematal animals. Preferredembodiments of the present invention include a mammalian PAO gene orgene product (e.g., cows, mice, humans, rats, pigs, monkeys, etc.).

As used herein the term “enzyme” refers to a protein which catalyseschemical reactions of other substances without itself being destroyed oraltered upon completion of the reactions. Enzymes are divided into sixmain groups: oxidoreductases, transferases, hydrolases, lyases,isomerases and ligases. The term “enzymatic activity” refers to thecatalytic activity of an enzyme or the activity by which the rate of abiochemical reaction is increased without altering the nature or thedirection of the reaction. In preferred embodiments of the presentinvention the term “enzyme” is used in reference to mammalian PAOs.

The terms “polyamine oxidase” and “PAO” refer to an enzyme whichcatalyzes the oxidative cleavage of the N¹-acetylated polyaminessubstrates such as N¹-acetyl-spermidine (N¹-acetyl-Spd),N¹-acetyl-spermine (N¹-acetyl-Spm), or other polyamines to produce ashortened polyamine or diamine, hydrogen peroxide and3-acetamidopropanal. The term “substrate” refers to a substance uponwhich an enzyme acts.

As used herein, the terms “mammalian peroxisomal acetylpolyamine oxidaseinhibitor” “PAO inhibitor” “APAO inhibitor” and “oxidase inhibitor”refer to any compound which can be used to reduce activity of amammalian peroxisomal acetylpolyamine oxidase. Compounds which reduceAPAO activity can be identified using the methods disclosed herein inExample 5. In some embodiments, the term oxidase inhibitor refers to butis not limited to synthalin and N-(3-aminopropyl)-1,10 decanediamine.Some preferred oxidase inhibitors are contemplated to selectively reducethe amine oxidizing activity of peroxisomal acetylpolyamine oxidases (asopposed to also inhibiting the activity of cytosolic polyamineoxidases).

The term “reducing” as used herein in reference to inhibition ofmammalian peroxisomal acetylpolyamine oxidase activity refers toconditions suitable to effect a decrease in oxidase activity of at leasttwo-fold as defined by k_(cat) or the “apparent” k_(cat), which isexpressed as μmoles (micromoles) of substrate oxidized per min per mg ofenzyme ( i.e., μmol min⁻¹ mg⁻¹, where 1 Unit of activity=1 μmol min⁻¹mg⁻¹). The values of k_(cat) or the “apparent” k_(cat) are alsotypically expressed as units of sec⁻¹. The term “apparent” indicatesthat the kinetic parameter, k_(cat), was determined at a concentrationof the substrate (e.g., O₂) that is subsaturating (e.g., at aconcentration that is not at least several times the value of theMichaelis constant for the substrate). In preferred embodiments, thereduction is at least five-fold, more preferably at least ten-fold, andmost preferably at least 100-fold.

As used herein, the term “vector” refers to any nucleic acid moleculethat can incorporate foreign DNA and transfer it from one cell toanother. Vectors are often derived from plasmids, bacteriophages, orplant or animal viruses. Similarly, the term “expression vector” refersto a recombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression (e.g.,transcription and translation) of the operably linked coding sequence ina particular host organism.

The term “plasmid” as used herein, refers to a small, independentlyreplicating, piece of DNA. Similarly, the term “naked plasmid” refers toplasmid DNA devoid of extraneous material typically used to affecttransfection. As used herein, a “naked plasmid” refers to a plasmidsubstantially free of calcium-phosphate, DEAE-dextran, liposomes, and/orpolyamines.

As used herein, the term “expression” refers to the process by which agene's coded information is converted into an operable structure such asan mRNA and/or a protein molecule. Thus, expressed genes are those thatare transcribed into mRNA and then translated into protein, as well asthose that are transcribed into RNA but not translated into protein(e.g., transfer and ribosomal RNAs).

The term “promoter” as used herein, refers to a DNA nucleotide sequencethat when attached to an RNA polymerase molecule, will initiatetranscription. Bacterial promoters utilized in some embodiments of thepresent invention include the T7 and trc promoters. The trc promoter isa hybrid promoter derived from the trp and lac promoters. The term“operator” refers to the site of repressor binding on a DNA molecule. Insome embodiments of the present invention the lac operator is employed.

As used herein, the term host cell refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The term “transgene” as used herein, refers to a foreign gene that isplaced into an organism by introducing the foreign gene into newlyfertilized eggs or early embryos. The terms “foreign gene” and“exogenous gene” refers to any nucleic acid (e.g., gene sequence) thatis introduced into the genome of an animal by experimental manipulationsand may include gene sequences found in that animal so long as theintroduced gene does not reside in the same location as does thenaturally-occurring, “endogenous” gene.

The term “transformed host cell” refers to the genetic modification of acell by incorporation of free DNA. In preferred embodiments, thetransformed host cell is a bacterial cell. “Transformation” of bacteriais typically brought about by heat or osmotic shock, electroporation orconjugation with another bacterial species.

The terms “bacteria” and “bacterial” as used herein, refer toprokaryotic organisms (e.g., Archebacteria, Eubacteria, Cyanobacteria).In preferred embodiments, the term “bacteria” refers to Eubacteria,which can be further subdivided on the basis of their staining usingGram stain (e.g., gram-positive and gram-negative).

As used herein, the term “prokaryote” refers to organismsdistinguishable from “eukaryotes.” It is intended that the termprokaryote encompass organisms that exhibit the characteristicsindicative of prokaryotes, such as possessing a single, circularchromosome, the lack of a true nucleus, the lack of membrane-boundorganelles, and other molecular characteristics indicative ofprokaryotes.

As used herein, the term “eukaryote” refers to organisms distinguishablefrom “prokaryotes.” It is intended that the term eukaryote encompass allorganisms with cells that exhibit the usual characteristics ofeukaryotes such as the presence of a true nucleus bounded by a nuclearmembrane within which reside the chromosomes, the presence ofmembrane-bound organelles, and other characteristics commonly observedin eukaryotic organisms.

The term “recombinant DNA” refers to a DNA molecule that is comprised ofsegments of DNA joined together by means of molecular biologytechniques. Similarly, the term “recombinant protein” refers to aprotein molecule that is expressed from recombinant DNA.

The term “fusion protein” as used herein refers to a protein formed byexpression of a hybrid gene made by combining two gene sequences.Typically this is accomplished by cloning a cDNA into an expressionvector in frame with an existing gene. The fusion partner may act as areporter (e.g., βgal) or may provide a tool for isolation purposes(e.g., polyhistidine).

Suitable systems for production of recombinant proteins include but arenot limited to prokaryotic (e.g., Escherichia coli), yeast (e.g.,Saccaromyces cerevisiae), insect (e.g., baculovirus), mammalian (e.g.,Chinese hamster ovary), plant (e.g., safflower), and cell-free systems(e.g., rabbit reticulocyte).

As used herein, the term “affinity tag” refers to a short amino acidsequence, engineered into the sequence of a recombinant protein, to makeits purification easier. Examples of the affinity tags which are knownto the skilled person include but are not limited to (His)₆ orpolyhistidine, Myc, FLAG, hemagglutinin, glutathione-S-transferase(GST), and a maltose-binding protein (MBP) tag. These protein tags canbe located N-terninally, C-terminally and/or internally. In particular,the terms “his tag” and “polyhistidine tag” refer to the linear array ofsix histidine residues added to the amino or carboxy terminus of arecombinant protein, in order to easily purify the recombinant proteinvia metal affinity chromatography using a nickel-chelating resin or byuse of polyhistidine-specific antibodies.

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense, and are meant to include aspecimen or culture. These terms encompasses all types of samplesobtained from humans and other mammals, including but not limited tobody fluids such as urine, blood, fecal matter, cerebrospinal fluid(CSF), semen, saliva, and wound exudates, as well as solid tissue.However, these examples are not to be construed as limiting the sampletypes applicable to the present invention.

As used herein, the term “patient” and “subject” refer to a mammal whois a candidate for receiving medical treatment. In some embodiments, thesubject is an individual suspected of having cancer, or havingexperienced a traumatic brain injury, or stroke.

The term “pathology” refers to the anatomic and/or physiologicaldeviations from the normal that constitute a disease.

In the present invention, “cancer” refers to a malignant tumor whosecells have the properties of endless replication, loss of contactinhibition, invasiveness and the ability to metastasize and whoseresult, generally, if left untreated, is fatal.

As used herein, the term “traumatic brain injury” refers to a physicalwounding suffered by the central nervous system and is characterized byblood-brain-barrier breakdown, marked edema formation, gliosis, andneuronal necrosis.

In the present invention, “stroke” refers to a cerebrovascular accidentcharacterized by a sudden loss of consciousness, often with resultingparalysis, caused by hemorrhage into the brain, either due to blockageof blood flow to the brain by an embolus or thrombus, or due to therupture of an artery exterior to yet supplying the brain, causing a lossof blood supply to the brain.

The term “control” refers to subjects or samples which provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to animals, which receive a mocktreatment.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that it isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

The terms “high stringency conditions” and “stringent conditions” whenused in reference to nucleic acid hybridization comprise conditionsequivalent to binding or hybridization at 42° C. in a solutionconsisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/lEDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5× Denhardt's reagent and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 0.1×SSPE, 1.0% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH2PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 g/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

As used herein, the term “Northern blot” refers to methods fortransferring denatured RNA onto a solid support for use in a subsequenthybridization assay. Total RNA or polyA-enriched RNA is typicallyelectrophoresed in an agarose gel, transfered to a membrane and probedwith a radioactively-labeled DNA or RNA fragment to detect specific RNAsequences. Northern blots are routinely used in the art (See, e.g.,Thomas, Proc Natl Acad Sci USA 77:5201-5205 [1980]; and Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,New York [1994]).

The term “Southern blot,” as used herein, refers to methods fortransferring denatured DNA, which has been fractionated by agarose gelelectrophoresis, onto a solid support, for use in a subsequenthybridization assay. These methods typically entail the digestion ofgenomic DNA with a suitable restriction enzyme prior to agarose gelelectrophoresis, transfer of the DNA to a membrane and incubation with aradioactively-labeled DNA or RNA fragment for detection of specific DNAsequences. Southern blots are routinely used in the art (See, Southern,J Mol Biol 98:503-517 [1975]; and Ausubel et al., supra [1994]).

As used herein, the term “polymerase chain reaction (PCR)” refers to amethod for increasing the concentration of a segment of a targetsequence in a DNA mixture without cloning or purification (See, e.g.,U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporatedby reference). This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.” When the template is RNA, a reverse transcription(RT) step is completed prior to the amplification cycles. Thus, thisvariation is termed “RT-PCR.”

The terms “primers” and “primer pair” as used herein, refers torelatively short pre-existing polynucleotide chains to which newdeoxyribonucleotides can be added by a DNA polymerase.

As used herein the term “electrophoresis” refers to a method ofseparating molecules in a mixture (e.g., DNA or RNA fragments, orproteins). Specifically, an electric current is passed through a mediumcontaining the mixture, and each molecule travels through the medium ata different rate, thereby causing the molecules to separate based upontheir size and charge. Agarose gels are typically used forelectrophoresis of nucleic acids, while polyacrylamide gels are commonlyused for electrophoresis of proteins. “Agarose gels” are produced from alinear galactan purified from agar that forms a gel when it is heatedand cooled. The term “ethidium bromide” as used herein, refers to a dyeused to stain nucleic acids, which fluoresces when exposed toultraviolet light.

The term “antibody” refers to polyclonal and monoclonal antibodies.Polyclonal antibodies which are formed in the animal as the result of animmunological reaction against a protein of interest or a fragmentthereof, can then be readily isolated from the blood using well-knownmethods and purified by column chromatography, for example. Monoclonalantibodies can also be prepared using known methods (See, e.g., Winterand Milstein, Nature, 349, 293-299 [1991]). As used herein, the term“antibody” encompasses recombinantly prepared, and modified antibodiesand antigen-binding fragments thereof, such as chimeric antibodies,humanized antibodies, multifunctional antibodies, bispecific oroligo-specific antibodies, single-stranded antibodies and F(ab) orF(ab)₂ fragments. The term “reactive” in used in reference to anantibody indicates that the antibody is capable of binding an antigen ofinterest. For example, a PAO-reactive antibody is an antibody that bindsto PAO or to a fragment of PAO.

The term “specific binding” when used in reference to the interactionbetween an antibody and an antigen describes an interaction that isdependent upon the presence of a particular structure (i.e., theantigenic determinant or epitope) on the antigen. In other words, theantibody recognizes a protein structure unique to the antigen, ratherthan binding to all proteins in general (i.e., non-specific binding).

As used herein, the term “immunoassay” refers to any assay that uses atleast one specific antibody for the detection or quantitation of anantigen. Immunoassays include, but are not limited to, Western blots,ELISAs, radio-immunoassays, immunofluorescence assays,immunohistochemistry and flow cytometry.

The terms “immunoblot” “Western blot” and “Western” refer to methods ofdetecting a specific protein or proteins in a complex protein mixturesuch as a cell extract or lysate. These methods, which are well known inthe art (See, e.g., Towbin et al., Proc Natl Acad Sci USA 76:4350-4354[1979]; and Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc., New York [1994]), involvefractionating the protein mixture by SDS-polyacrylamide gelelectrophoresis, transferring the separated proteins onto a solidsupport such as nitrocellulose and detecting the protein(s) of interestby with an antibody. The bound primary antibody can be visualized by theuse of a secondary antibody conjugated to an enzyme which produces asignal in the presence of a suitable substrate.

As used herein, the term “ELISA” refers to enzyme-linked immunosorbentassay. Numerous ELISA methods and applications are known in the art, andhave already been described in detail elsewhere (See, e.g., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press [1988]; andAusubel et al. (eds.), Current Protocols in Molecular Biology, Ch. 11,John Wiley & Sons, Inc., New York [1994]). The term “ELISA” includes butis not limited to direct ELISA, indirect ELISA, sandwich ELISA and cellELISA methods. For instance, a cell sample of interest is coated ontothe bottom of a well of a 96-well microtiter plate, and under suitableconditions the sample is contacted with a PAO-reactive antibody, whosebinding is then visualized by contact with a peroxidase-conjugated goatanti-mouse serum and a calorimetric substrate.

The terms “inimunofluorescent analysis” and “IFA” refer to microscopymethods in which a fluorescent-labelled antibody (e.g., a PAO-reactivemonoclonal antibody conjugated to fluorescein) is used to detect thepresence or determine the location of the corresponding antigen (e.g.,hPAO in permeabilized hepatocytes) using a fluorescence microscope(e.g., microscope with ultraviolet light source).

As used herein, the term “immunohistochemistry” refers to microscopymethods in which location of an antigen of interest is visualized byusing a labelled antibody as a detection agent. In some embodiments themicroscope has a visible light source and the antibody of interest islabelled with an enzyme suitable for use in a calorimetric reaction.

The terms “flow cytometry” and “FACS analysis” refer to methods ofmeasuring fluorescence from a suspension of stained cells (e.g., cellsthat have been placed in contact with a PAO-reactive antibody) flowingthrough a narrow orifice, usually in combination with one or two lasersto activate the dyes (e.g., fluorochrome bound to either primary orsecondary antibody).

The term “reporter molecule” as used herein, refers to molecules such asenzymes and fluorochromes which are suitable for use as tools fordetection of an antigen of interest (e.g., PAO). Appropriate reporterenzymes include but are not limited to enzymes that can be utilized incolorimetric reactions, including but not limited to alkalinephosphatase and horse-radish peroxidase. As used herein, the terms“fluorochrome” and “fluorescent dye” refer to compounds which have theability to emit light of a certain wavelength when activated by light ofanother wavelength. In some embodiments, the term “fluorochrome”includes but is not limited to fluorescent compounds such asfluorescein, phycoerythrin, Texas red, and rhodamine.

As used herein, the term “kit” is used in reference to a combination ofreagents and other materials which facilitate sample analysis. In someembodiments, the immunoassay kit of the present invention includes asuitable capture antibody, reporter antibody, antigen, detectionreagents and amplifier system. Furthermore, in other embodiments, thekit includes, but is not limited to, components such as apparatus forsample collection, sample tubes, holders, trays, racks, dishes, plates,instructions to the kit user, solutions or other chemical reagents, andsamples to be used for standardization, normalization, and/or controlsamples.

DESCRIPTION OF THE INVENTION

This invention relates to compositions and methods for the treatment ofpathologies associated with intracellular polyamine dysregulation. Inparticular, the present invention provides compositions and methodsinvolving mammalian N¹-acetyl-polyamine oxidase (APAO or PAO) to treatcancer, cell damage, tissue damage caused by ischemia and reperfusion,inflammation, traumatic brain injury, stroke, and tissue developmentaldisorders. Methods for diagnosis and prognosis of cancer and otherdiseases are also provided by the present invention.

Functions of Polyamine Oxidase (PAO)

PAO has been implicated in many physiological functions.Inmnunoregulatory roles have been proposed for PAOs in pregnancy (Morganand Illei, Br. Med. J. 280:1295-1297 [1980]), and in some autoimmunedisease (Flescher et al., J. Clin. Invest. 83:1356-1362 [1989]).Oxidized polyamines have antimicrobial, antiviral (Bachrach, inPolyamines in Biology and Medicine, Morris and Marton (eds.) MarcelDekker, New York, pp.151-168 [1981], antifungal (Levitz et al., Antonievan Leeuwenhoek 58:107-114 [1990]), and antiparasitic properties(Rzepczyk et al. Infect. Immun. 43:238-244 [1984]). Moreover, PAOs havebeen implicated in apoptotic processes in both animal (Parchmnet, inPolyamine in Cancer:Basic Mechanisms and Clinical Approaches, Nishioka(ed.) R. G. Landes, Austin pp. 99-150 [1996]) and plant cells.

The intracellular level of polyamines is strictly regulated by themodulation of the activities of enzymes that are involved in thepathways for polyamine biosynthesis and degradation, as well as systemsinvolved in their transportation (Pegg, Biochem J. 234:249-62 [1986],Casero and Pegg, FASEB. J. 7:653-61 [1993]). The first enzyme forpolyamine synthase is ornithine decarboxylase (ODC), which catalyses theconversion of ornithine into putrescine (Put). Subsequently Put isconverted into spermidine (Spd) by Spd synthase, and Spd is convertedinto Spermine (Spm) by Spm synthase (Pegg, Biochem. J. 234:249-62[1986]; Seiler et al., Biochem. J. 225:219-226 [1985]; and Tabor andTabor, Annu. Rev. Biochem. 53:749-790 [1984]). Polyamine degradationoccurs via a polyamine catabolic pathway that requires thespermine/spermidine N¹-acetyltransferase (SSAT) and PAO. In thispathway, Spm and Spd are acetylated first by SSAT in the cytoplasmicmatrix (Pegg, Biochem. J. 234:249-62 [1986]; Pegg and McCann, Am. J.Physiol. 245:C212-221 [1981], van den Munckhofet al., J. Histochem.Cytochem. 43:1155-1162 [1995]). Next, N¹-acetyl-Spm and N¹-acetyl-Spdare oxidized by PAO to Spd and Put, respectively, within peroxisomes(van den Munckhof et al., supra [1995]). Thus, PAO is an integral partof the polyamine interconversion cycle, a major regulatory system thatcontributes to maintaining polyamine homeostasis of animal cells(Cipolla et al., Urol. Res. 24:93-98 [1996]). PAO activity is ratherhigh when compared with activities of the biosynthetic enzymes. Thus,PAO is deemed important in regulating tissue levels of the polyamine(Shipe et al., CRC Critical Rev. in Clinical Lab Science [1981]). Alsoas part of the catabolic pathway, spermine oxidase (Wang et al., supra[2001]; Murray-Stewart et al., supra [2002]; Vujcic, et al., supra[2002]; and in International Patent Application No. WO 02/100884)converts Spm to Spd. PAO can also convert Spm to Spd, albeitinefficiently.

The elevated levels of N¹-acetylated polyamines observed in a variety oftumors, is associated with reduced PAO activity, suggesting that aproduct of polyamine oxidation may regulate cell replication (Morgan,Biochem. Soc. Trans. 13:322-326 [1985]; Bachrach, supra [1981]). Forinstance, the loss of PAO activity is directly related to the size andgrade of human breast tumors (Wallace et al., Clin. Cancer Res.6:3657-3661 [2000]), as the N¹-acetyl-Spm:Spd ratio gradually increasesfrom stage I to stage IV breast cancer tumors (Lee et al., Cancer Lett.133:47-56 [1998]). Similarly, the N¹-acetylated polyamine levels aremuch higher and PAO activity much lower in human colorectal cancertumors than in neighboring tissue (Linsalata, Cavallini and Di Leo,Anticanc. Res. 17:3757-3760 [1997]). Numerous other studies furtherdemonstrate the intimate involvement of PAO in cancer (See, e.g., Hu andPegg, Biochem. J. 328:307-316 [1997]; Kramer et al., Cancer Res.59:1278-1286 [1999]; Bergeron et al., J. Med. Chem. 43:224-235 [2000];Mank-Seymour et al., supra [1998]; Lindsay, and Wallace, supra [1999];and Chopra and Wallace, supra [1998]).

An unidentified human tumor-suppressor gene has been localized to thesame region of Chromosome 10 as the hpao gene (Kim et al., Oncogene17:1749-1753 [1998]; Albarosa et al., Genomics 41:345-9 [1997]; Chernovaet al., Oncogene 20:5378-5392 [2001]; and Lee et al., Mamm. Genome12:157-162 [2001]). Deletions or disruptions in this region have beenfound in small cell lung cancer (Kim et al., supra [1998]), bladdercancer (Kagan et al., Oncogene 16:909-913 [1998]), prostate cancer(Komiya et al., Genes, Chromosomes & Cancer 17:245-253 [1996]), braintumors (Albarosa et al., supra [1997]; and Lee et al., supra [2001]),and glioblastoma cells (Chernova et al., supra [2001]).

The discovery of gene deletion and transcriptional variants of a humanPAO gene, during the development of the present invention, indicate thatPAO plays a fundamental role in controlling cell growth. Thus, hpao iscontemplated to be a tumor-suppressor gene.

Molecular Biology and Biochemistry of PAO

PAO has been purified from rat, porcine and bovine liver (Seiler, supra[1995]; and Gasparian and Nalbandian, Biokhimiia 55:1632-1637 [1990]).PAO is a 56 kDa monomer, containing noncovalently bound FAD as theessential redox cofactor. Mammalian PAO has a narrow specificity fornatural substrates (Spm, N¹,N¹²-acetyl-Spm, N¹-acetyl-Spm andN¹-acetyl-Spd). A few irreversible inhibitors have been identifiedincluding MDL 72521 and MDL 72527 (Seiler, supra [1995]). PAOinactivates the anticonvulsant, milacemide [2-(n-pentylamino)acetamide],which is also a time-dependent irreversible inactivator of mitochondrialMAO-B (O'Brian et al., Biochem. Pharmacol. 47:617-623 [1994]). Inaddition, PAO oxidizes the antimalariavantiparasitic agent MDL 27695(N,N-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane) to aninactive form (Edwards et al., J. Med. Chem. 34:569-574 [1991]). Clearlythe action of PAO may reduce the efficacy of other drugs. Thus, it iscontemplated that a thorough understanding of the biochemical andkinetic properties of PAO provides the means for the design of drugsthat are less prone to inactivation by this oxidase.

Similar flavoproteins (e.g., glutathione reductase and the flavoproteinsubunit of the flavocytochrome c sulfite reductase) have been used tomodel the structure of MAO-A (Wouters and Baudoux, Proteins: Struct.Funct. Genet. 32:97-110 [1998]), or to select amino acyl residues ofMAO-A and MAO-B for site-specific mutagenesis (Zhou et al., J. Biol.Chem. 273:14862-14868 [1998]; and Kirksey et al., Biochemistry37:12360-12366 [1998]). However, prior to the development of the presentinvention, only the structures of the corn PAO and MAO-B were known(Binda et al., supra [1999]; and Binda et al., supra [2002]). Thus,little biochemical and structural information was available for PAO,prior to the development of the present invention.

In addition to the mammalian peroxisomal N¹-acetyl-polyamine oxidasegenes described herein, the gene sequences of several forms of PAO areknown; a so-called N¹-acetyl-Spd oxidase from Candida boidiniiperoxisomes (Nishikawa et al., FEBS Lett. 476:150-154 [2000]) a Spm/Spdoxidase from corn (Tavladoraki et al., FEBS Lett. 426: 62-66 [1998]), acytosolic human Spm oxidase (Wang et al., Cancer Res. 61:5370-5373[2001]; Murray-Stewart, et al., Biochem. J. 368:673-677 [2002]). and acytosolic murine Spm oxidase (GenBank Accession No. BC004831; Vujcic, etal., Biochem. J. 367:665-675 [2002]). The translated protein sequencesof all of these amine oxidases have similarities with the bovine PAO(bPAO), murine PAO (mPAO) and human (hPAO) protein sequences describedherein. In particular, they all have an easily identifiable FAD-bindingmotif near their N-termini. This, and other features of the primarystructures, indicate that these enzymes are members of a larger class ofamine oxidases, which includes: mitochondrial MAO-A and MAO-B, MAO-Nfrom Aspergillus niger (S chilling and Lerch, Biochim. Biophys. Acta1243:529-537 [1995]; and Sablin et al., Eur. J. Biochem. 253:270-279[1998]), putrescine oxidase from Micrococcus rubens (Ishizuka et al., J.Gen. Microbiol. 139:425-432 [1993]), and tyramine oxidase fromMicrococcus luteus (Roh et al., Biochem. Biophys. Res. Commun.268:293-297 [2000]). Other evidence indicates that these oxidases belongto an even larger class (e.g., superclass) of enzymes called the GR₂(glutathione reductase) superclass of flavoproteins (Dailey and Dailey,J. Biol. Chem. 273:13658-13662 [1998]; Dym and Eisenberg, Protein Sci10:1712-1728 [2001]). This superclass includes glucose oxidase,cholesterol oxidase, D-amino acid oxidase, sarcosine oxidase,p-hydroybenzoate hydroxylase, phenol hydroxylase, fumarate reductase,succinate dehydrogenase, glutathione reductase, protoporphyrinogenoxidase and phytoene desaturase.

As detailed below in the Examples, the genes for PAO from humans, miceand cattle were cloned during development of the present invention.Moreover, PAO expression in normal mouse and human tissues was examined.PAO expression was also assessed in the context of apoptosis induced byN¹-acetyl-Spm treatment of cultured cells. In addition, recombinanthuman and murine PAOs were produced in bacterial expression systems.With the known corn PAO structure as the template (Binda, et al., supra[1999]), the mPAO amino acid sequence was used to homology model thestructure of this mammalian oxidase.

Role of PAO in Development

Recycling of Put by intracellular PAO may be important for maturinganimals. PAO is scarce in the brain and liver of newborn rats, butincreases dramatically during postnatal development (Seiler, supra[1995]). Put does not easily pass through the blood brain barrier, andits half-life in the brain is longer than in other tissues. Thiseffectively isolates the brain's polyamine pool from the rest of thebody. Thus, PAO may be of paramount importance for the maintenance ofthe polyamine pool of the brain.

In adult rats, long-term inhibition of PAO by MDL 72527(N¹,N⁴-bis(2,3,-butadienyl)-butane-1,4-diamine) leads to elevated levelsof the N¹-acetylated polyamines, reduced Put and Spd levels in tissues,and a rise in the blood Spm levels. However, PAO inhibition had noapparent adverse effect on the MDL 72527-treated rats. Normal, matureanimals do not suffer from a loss of PAO-generated Put or a build-up ofthe N¹-acetylated polyamines, as these forms are excreted from the cell.Compensation for lost Put can be achieved by an increased ODC activity,and Put can be acquired readily from the diet. Any excess N¹-acetylatedpolyamines are transported in the circulation to the kidney forexcretion in urine. Apparently, under normal circumstances in matureanimals, the primary role of PAO is for the maintenance of intracellularpolyamine homeostasis (Morgan, Biochem. Soc. Trans. 26:586-591 [1998];and Seiler, supra [1995]).

Role of PAO in Cancer Growth

While very high intracellular levels of Put and the polyamines are toxicto normal cells, cancer cells have an increased demand for thesesubstances. MDL 72527 inhibition of PAO in tumor cells decreased the Putlevels of the cells and slowed tumor growth. This drug was a moreeffective antitumor agent when used in conjunction with the potent ODCinhibitor 2-(difluoro-methyl)ornithine (Seiler, supra [1995]). Polyaminedepletion is known to stimulate the immune system, therefore, a directimmune response may also contribute to the observed arrest in tumorgrowth.

Anti-cancer drugs cause increased expression of SSAT, which depletescancer cells of Spm and Spd. N¹,N¹¹-bis(ethyl)norspermine down-regulatesthe polyamine biosynthetic enzymes ODC and S-adenosylmethioninedecarboxylase, but dramatically up-regulates SSAT production (Hu andPegg, Biochem. J. 328:307-316 [1997]; and Kramer et al., Cancer Res.59:1278-1286 [1999]). In Chinese hamster ovary (CHO) cells,N¹,N¹¹-bis(ethyl)norspermine induced apoptosis. However, addition of thespecific PAO inhibitor, MDL 72521(N¹-methyl-N⁴-(2,3-butadienyl)-butane-1,4-diamine), prevented apoptosis(Hu and Pegg, supra [1997]). NCI H727 human female carcinoid cells haveviable ssat genes on both X chromosomes, in contrast to normal cellswhere only a single ssat allele is translated into active SSAT. Additionof the antitumor polyamine analog, N¹,N¹²-bis(ethyl)spermine BESpm), toa H727 cell culture led to high levels of SSAT mRNA and SSAT activity.Apparently, an inappropriate expression of both ssat genes caused thehigher sensitivity of the H727 tumor cells to the anticancer drug, BESpm(Mank-Seymour et al., supra [1998]). In both CHO cells and in femalecarcinoma cells, the hyper-expression of SSAT increased the productionof N¹-acetylated polyamines. The elevated N¹-acetylated polyamine levelin turn, resulted in higher PAO activity and apoptosis, indicating thatPAO-generated hydrogen peroxide and possibly pre-cytotoxic3-acetamidopropanal play important roles in apoptosis.

Etoposide, a topoisomerase II inhibitor, induced apoptosis in HL-60human promyelogenous leukemia cells (Lindsay and Wallace, Biochem. J.337:83-87 [1999]). While it was shown that alterations in polyamineoxidation did not initiate apoptosis, it was suggested that aPAO/SSAT-dependent cell death-generating cycle was slowly set in motion,peaking at approximately 48 hours. In particular, PAO oxidizedN¹-acetylated polyamines to produce hydrogen peroxide, stimulating SSATactivity (Chopra and Wallace, Biochem. Pharmacol. 55:1119-1123 [1998]).Increased SSAT activity lead to increased levels of the N¹-acetylatedpolyamines, resulting in higher PAO activity and increased hydrogenperoxide and 3-acetamidopropanal production.

Role of PAO in Traumatic Brain Injury and Cerebral Ischemia/Reperfusion

Cerebral trauma escalated polyamine oxidation via PAO, exacerbating theinjury. After inducing transient cerebral ischemia in rats,administration of the PAO inhibitor MDL 72527 lowered the reperfusioninjury volume (Dogan et al., J. Neurosurg. 90:1078-1082 [1999]).Similarly, rats inflicted with traumatic brain injury had less brainedema formation and delayed cellular damage when given MDL 72527 (Doganet al., J. Neurochem. 72:765-770 [1999]). PAO-generated 3-aminopropanal,rather than hydrogen peroxide, was identified as the culprit in ischemicneuronal and glial cell death (Ivanova et al., J. Exp. Med. 188:327-340[1998]; Ivanova et al., Pro. Natl. Acad. Sci. USA 99:5579-5584 [2002]).In fact, 3-aminopropanal is cytotoxic to cultured glial cells,fibroblasts, endothelial cells, and various transformed cell lines.There is also evidence to suggest that 3-aminopropanal plays a role inapoptosis during murine embryonic limb bud formation, and that it may beinvolved in tumor necrosis (Ivanova et al., supra [1998]; and Parchmentand Pierce, Cancer Res. 49:6680-6686 [1989]).

PAO Knock Out Animals and Transgenic Animals Expressing Exogenous PAOGenes, Mutants, and Variants Thereof

The present invention contemplates the generation of PAO-gene knock outanimals, and transgenic animals comprising an exogenous PAO gene orhomologs, mutants, or variants thereof. In preferred embodiments, theknock out and transgenic animals display an altered phenotype (e.g.,dysregulation of polyamine metabolism) as compared to wild-type animals.In some embodiments, the altered phenotype is the overexpression of mRNAfor a PAO gene as compared to wild-type levels of PAO expression. Inother embodiments, the altered phenotype is the decreased expression ofmRNA for an endogenous PAO gene as compared to wild-type levels ofendogenous PAO expression. Methods for analyzing the presence or absenceof such phenotypes include Northern blotting, mRNA protection assays,and RT-PCR. In other embodiments, the transgenic animals have a knockout mutation of the PAO gene. In still further embodiments, thetransgenic animals express a PAO variant or a truncated PAO.

The transgenic animals of the present invention find use in dietary anddrug screens. In some embodiments, the transgenic animals are fed testor control diets and the response of the animals to the diets isevaluated. In other embodiments, test compounds (e.g., a drug that issuspected of being useful to treat diseases contemplated to beassociated with dysregulation of polyamine metabolism such as cancer)and control compounds (e.g., a placebo) are administered to thetransgenic and control animals, and the effects are then evaluated.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes into a normal animal orpao-knock outanimal, for the production of transgenic animals. Different methods areused depending on the stage of development of the embryonal cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 picoliters (pl) of DNAsolution. The use of zygotes as a target for gene transfer has a majoradvantage in that in most cases the injected DNA will be incorporatedinto the host genome before the first cleavage (Brinster et al., Proc.Natl. Acad. Sci. USA 82:4438-4442 [1985]). As a consequence, all cellsof the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the foinder since 50% ofthe germ cells will harbor the transgene. U.S. Pat. No. 4,873,191describes a method for the micro-injection of zygotes; the disclosure ofthis patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a normal or pao-knockout non-human animal. In someembodiments, the retroviral vector is utilized to transfect oocytes byinjecting the retroviral vector into the perivitelline space of theoocyte (U.S. Pat. No. 6,080,912, incorporated herein by reference). Inother embodiments, the developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Janenich, Proc. Natl. Acad. Sci. USA73:1260 [1976]). Efficient infection of the blastomeres is obtained byenzymatic treatment to remove the zona pellucida (Hogan et al., inManipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. [1986]). The viral vector system used to introducethe transgene is typically a replication-defective retrovirus carryingthe transgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al., Nature 298:623 [1982]).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells which form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germiline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal., supra [1982]). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application No. WO 90/08832 [1990], andHaskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells which haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or to create deletion mutants (e.g., mutants inwhich various PAO domains are deleted). Methods for homologousrecombination are described in U.S. Pat. No. 5,614,396, incorporatedherein by reference.

Drug Screening Using PAO

The present invention also provides methods and compositions for usingPAO as a target for screening drugs that can alter polyamine metabolism.

One technique for drug screening provides high throughput methods foridentifying compounds having suitable binding affinity to PAO peptidesand is described in detail in WO 84/03564, incorporated herein byreference. Briefly, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The peptide test compounds are then reacted with PAOpeptides and washed. Bound PAO peptides are then detected by methodswell known in the art.

Another technique uses PAO antibodies, generated using methods known inthe art. Such antibodies capable of specifically binding to PAO peptidescompete with a test compound for binding to PAO. In this manner, theantibodies can be used to detect the presence of any peptide that sharesone or more antigenic determinants of the PAO peptide.

In some embodiments of the present invention, compounds are screened fortheir ability to inhibit the binding of a labeled substrate to PAO. Anysuitable screening assay may be utilized, including, but not limited to,those described herein. The present invention also contemplates manyother means of screening compounds. The examples provided above arepresented merely to illustrate a range of techniques available. One ofordinary skill in the art will readily appreciate that many otherscreening methods can be used.

In particular, the present invention contemplates the use of cell linestransfected with PAO and variants or mutants thereof for screeningcompounds for activity, and in particular to high throughput screeningof compounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in reporter gene assays that monitor cellularresponses at the transcription/translation level. In still furtherembodiments, the cells can be used in cell proliferation assays tomonitor the overall growth/no growth response of cells to externalstimuli.

In some assays, the host cells are preferably transfected as describedabove with vectors encoding PAO or variants or mutants thereof. The hostcells are then treated with a compound or plurality of compounds (e.g.,from a combinatorial library) and assayed for the presence or absence ofa response. It is contemplated that at least some of the compounds inthe combinatorial library can serve as agonists, antagonists,activators, or inhibitors of the protein or proteins encoded by thevectors. It is also contemplated that at least some of the compounds inthe combinatorial library can serve as agonists, antagonists,activators, or inhibitors of protein acting upstream or downstream ofthe protein encoded by the vector in a signal transduction pathway.

The cells are also useful in reporter gene assays. Reporter gene assaysinvolve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i. e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product.

Pharmaceutical Compositions Containing PAO, Analogs and Inhibitors

The present invention further provides pharmaceutical compositions whichmay comprise all or portions of PAO polynucleotide sequences, PAOpolypeptides, inhibitors or antagonists of PAO bioactivity, includingantibodies, alone or in combination with at least one other agent, suchas a stabilizing compound, and may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states. Peptides can be administered to thepatient intravenously in a pharmaceutically acceptable carrier such asphysiological saline. Standard methods for intracellular delivery ofpeptides can be used (e.g. delivery via liposome). Such methods are wellknown to those of ordinary skill in the art. The formulations of thisinvention are useful for parenteral administration, such as intravenous,subcutaneous, intramuscular, and intraperitoneal. Therapeuticadministration of a polypeptide intracellularly can also be accomplishedusing gene therapy as described above.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Suitable routes may, for example, include oral or transmucosaladministration; as well as parenteral delivery, including intramuscular,subcutaneous, intrarnedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of PAO may be that amount that suppresses apoptosis.Determination of effective amounts is well within the capability ofthose skilled in the art, especially in light of the disclosure providedherein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For PAO polynucleotides, polypeptides or inhibitors,conditions indicated on the label may include treatment of conditionsrelated to apoptosis.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels particularly murine models) to achieve a desirable circulatingconcentration range that adjusts PAO levels.

A therapeutically effective dose refers to that amount of PAO or PAOinhibitor which ameliorates symptoms of the disease state. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and additional animalstudies can be used in formulating a range of dosage for human use. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Those skilled in theart will employ different formulations for PAO than for the inhibitorsof PAO. Administration to the bone marrow may necessitate delivery in amanner different from intravenous injections.

Experimental

The following Examples serve to illustrate certain preferred embodimentsof the present invention and are not to be construed as limiting thescope thereof. All chemicals were of reagent grade or better, andpurchased from common vendors.

In the experimental disclosure below, the following abbreviations apply:FAD (flavin adenine dinucleotide); ODC (ornithine decarboxylase); SSAT(CoenzymeA: spermidine/spermine-N¹-acetyltransferase); PAO and pao(polyamine oxidase and its gene, respectively); bPAO and bpao (bovinePAO and its gene, respectively); mPAO and mpao (murine PAO and its gene,respectively); hPAO and hpao (human PAO and its gene, respectively);cPAO (corn/maize PAO); MAO (mitochondrial monoamine oxidase); Spm(spermine); Spd (spermidine); Put (putrescine); cDNA (complementaryDNA); EST (expressed sequence tag); kb (kilobases); bp (basepairs); HPLC(high performance liquid chromatography); PDB (Protein Data Bank); PCR(polymerase chain reaction); RACE (rapid amplification of cDNA ends);SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); MW(molecular weight); Da (daltons); kDa (kilodaltons); eq (equivalents); μ(micron); M (Molar); μM (micromolar); mM (millimolar); N (Normal); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); kg(kilograms); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); L (liters); mL (milliliters); μL (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nM (nanomolar); ε(extinction coefficient); ° C. (degrees Centigrade); OD (opticaldensity); UV (ultraviolet); psi (pounds per square inch); hydroxyapatite(HAP); N-[2-hydroxyethyl]piperazine-N¹-[2-ethanesulfonic acid] (HEPES);isopropyl thio-β-D-galactopyranoside (IPTG); kan (kanamycin); LB (LuriaBertani); 3-(N-morpholino)propane (MOPS); PBS (phosphate bufferedsaline); hrs (hours); RT (room temperature); Avestin (Avestin Inc.,Ottawa, Ontario, CANADA); BIO 101 (Qbiogene, Carlsbad, Calif.); Bio-Rad(Bio-Rad Laboratories, Hercules, Calif); Boehringer (Boehringer Manheim,Germany); Clontech (Clontech Laboratories, Palo Alto, Calif.); HewlettPackard (Hewlett Packard, Palo Alto, Calif.); IMAGE (IMAGE Consortium,Lawrence Livermore National Laboratory, Livermore, Calif.); Incyte orGenome Systems (Incyte Genomics Inc., Palo Alto, Calif.); Invitrogen(Invitrogen Corporation, Carlsbad, Calif.); Amicon or Millipore(Millipore Corp., Bedford, Mass.); Novagen (Novagen, Inc., Madison,Wis.); Operon (Operon Technologies Inc., Alameda, Calif.); Origin(Origin Technology, Inc., Rockville, Md.); Pharmacia (Amersham PharmaciaBiotech Inc., Piscataway, N.J.); Qiagen (Qiagen Inc., Valencia, Calif.);Sigma (Sigma Aldrich Corporation, St. Louis, Mo.); Spectrum (SpectrumSoftware Associates, Chapel Hill, N.C.); and Stratagene (Stratagene,Inc., La Jolla, Calif.).

EXAMPLE 1 Purification of bPAO and Microsequencing

In this example, the purification and amino acid sequencing of bPAOpeptides is described. Fresh bovine livers were obtained at a localslaughterhouse. Immediately after removal from the animal, they werecovered with ice and were transported to the laboratory within 1 hr.Following a published procedure (Gasparian, Biokhimiia 60:1632-1636[1995]), bPAO was purified from the fresh liver, or tissue that had beenstored at −70° C. as 1-inch cubes. Approximately 1 mg of nearly purebPAO was obtained from 1 kg of tissue. The enzyme was purified furtheron a 10% Tris-HCl SDS Ready Gel (Bio-Rad), then electro-transferred ontoan Immobilon-P^(sQ) membrane (Millipore), and Coomassie blue-stained.The membrane was submitted to the Biomolecular Resource Center(University of California, San Francisco, Calif.) for N-terminalsequence analysis by an automated Edman degradation procedure. SDS-PAGEpurified bPAO yielded the N-terminal amino acid sequence:EAEAPGRGPRVLVVGGGIAGL (SEQ ID NO:7). Purified bPAO was electrophoresedagain as above, and the Coomassie-blue stained bPAO band was excised andsubjected to an in-gel tryptic digest at the Protein Sequencing Center(State University of New York, Brooklyn, N.Y.). Two major peptides werepurified and sequenced, yielding the internal amino acid sequencesSEHSFGGVVEVGAHWIHGPS (SEQ ID NO:8) and LMTLWDPQAQWPEPR (SEQ ID NO:9).The segments of the bPAO amino acid sequence that correspond to theshared motif of the FAD-containing protein superfamily are underlined(Dailey and Dailey, J. Bio. Chem. 273:13658-13662 [1998]).

EXAMPLE 2 Cloning and Sequencing bPAO, mPAO, and hPAO

In this example, methods used to determine the mammalian PAO DNAsequences are described. The three bPAO peptide sequences described inExample 1, were used to search the GenBank and GenBank EST (expressedsequence tag) databases. Two mouse EST sequences that coded for an aminoacid sequence 85% identical to that of SEQ ID NO:8 were identified(e.g., GenBank Accession Nos. AA437705 and AI098814, corresponding toIMAGE Nos. 819909 and 1482295, respectively). A bacterial strain (DH10B)containing the cDNA clone of GenBank Accession No. AA437705, which isflanked by SP6 and T7 promoters of the vector pSPORT1, was obtained fromIncyte and sequenced. A 968 bp Xba I/Sal I fragment (mpao1) was excisedfrom the purified plasmid, and labeled with ³²P-dATP (Pharmacia) byusing a random primed DNA labeling kit (Boehringer). A λgt10 mouse 17day embryo cDNA library (CLONTECH) with a titer of 1.1×10¹⁰ pfu/mL, anda Uni-ZAP XR bovine liver cDNA library (Stratagene) with a titer of2.2×10¹⁰ pfu/mL were screened with the radiolabeled mpao1 probe usingestablished methods (See, e.g., Sambrook et al., Molecular Cloning:ALaboratory Manual, second edition (Cold Spring Harbor Laboratory Press,New York) [1989]). About 2.7×10⁶ plaques for a bovine liver cDNAlibrary, and about 2.5×10⁶ plaques for a mouse embryo cDNA library werescreened. Seventeen positive bovine and sixteen positive mouse plaquesisolated from the primary screening, were re-screened. The finalpositive clones contained mpao cDNA inserts of different lengths, asconfirmed by Southern blotting (Sambrook et al., supra [1989]), usingP³²-labeled mpao1 as a probe. Several positive plaques with large paofragments were isolated, subcloned and sequenced. Excision of thepBluscript phagemid from the Uni-ZAP XR vector was carried out accordingto the manufacturer's instructions (Stratagene). The cDNA insertscontained in the pBluscript phagemids were sequenced using the T3 and T7flanking primers. A 1.6 kb fragment of bpao (bpao1), coding for most ofthe C-terminal portion of bPAO, was isolated. The 5′-end of the fragmentcoded for a sequence that exactly matched the N-terminal portion of SEQID NO:8, while a region near the 3′-end coded for a protein sequenceexactly matching the C-terminal end of SEQ ID NO:9, which was deduced tobe the C-terminus of the enzyme. The partial bPAO cDNA (SEQ ID NO:1) andprotein (SEQ ID NO:2) sequences are shown in FIG. 2.

DNA isolated from one plaque of the mouse λgt10 library, was treatedwith Sal I and cloned into the Sal I sites of pUC19 to give the plasmid,pUC19-MPAO1, which was used for double-stranded sequencing. The missing5′-segment of mpao was obtained by using the 5′-RACE PCR method usingmouse 17-day embryo Marathon Ready cDNA (CLONTECH) as the template, andusing a SMART RACE™ PCR cDNA Synthesis Kit (CLONTECH) for the PCRreactions. The primers used for 5′-RACE PCR include a mpao gene-specificantisense primer, mpao1R (Operon) spanning nucleotides 333 to 309 ofmpao (5′-GTTCTCTTCC GATAATTCTT TCTCC-3′, SEQ ID NO:10), and the CLONTECHAP1 universal sense adaptor primer which is specific for the MarathonReady cDNA template. The parameters used for PCR were as follows; 5cycles for 30 sec at 94° C. and 3 min at 72° C., 5 cycles for 30 sec at94° C. and 3 min at 70° C., and 30 cycles for 20 sec at 94° C. and 3 minat 68° C. Using a 50-fold dilution of the resulting PCR product astemplate, and AP2 (an AP1 nested primer) and mpao1R as primers, a secondPCR reaction was carried out using the same conditions. The resulting˜400 bp cDNA fragment, was isolated from an 1% agarose gel using the BIO101 Gene Clean Kit, and sequenced. The PCR product, along with mpao1,yielded a 1.7 kb section of mpao (mpao2) coding for an amino acidsequence that is extremely similar to the predicted amino acid sequenceof bpao1. The complete mPAO cDNA (SEQ ID NO:3) and protein (SEQ ID NO:4)sequences are shown in FIG. 3.

Subsequently, the mPAO cDNA sequence was used to screen the GenBank ESTdatabase for human entries. Several homologous human EST sequences wereidentified including an infant brain EST (GenBank Accession No. H05647).The cDNA clone corresponding to the human EST sequence was obtained fromGenome Systems. The sequence derived from this human EST had an openreading frame of 319 amino acids. This C-terminal amino acid sequencewas 83% identical to the aligned mPAO sequence. In order to isolate alarger portion of the human PAO cDNA, 5′-RACE PCR was used with a humanliver cDNA library as a template (Clontech). This strategy was usedsuccessfully to obtain additional 5′ coding and 3′ non-coding hPAOsequence. The remaining N-terminal amino acid sequence was obtained froma second human EST (GenBank Accession No. AW662266). The complete hPAOcDNA (SEQ ID NO:5) and protein (SEQ ID NO:6) sequences are shown in FIG.4, and an alignment of all three mammalian PAO protein sequences isshown in FIG. 5. The PRL motif at the C-terminus of the three mammalianPAO sequences is a putative peroxisomal transport sequence (Gould, J.Cell Bio. 108:1657-1664 [1989]).

The hPAO cDNA sequence was used to BLAST search the human genomedatabase from the National Center for Biotechnology Information website.The entire hpao sequence (contained in GenBank Accession No.AL360181.37) was found to map as 7 exons (See, FIG. 6 and SEQ ID NO:14,where base No. 2840 of this sequence corresponds to base No. 83941 ofGenBank Accession No. AL360181.31). For SEQ ID NO:14, Exon I=bases2840-3072, Exon II=bases 3574-4058, Exon III=bases 5033-5232, ExonIV=bases 7533-7785,Exon V=bases 12539-12641,Exon VI=bases13163-13320,andExon VII=bases 14885-15266. The hpao gene mapped near theterminus of the q arm of chromosome 10, at cytogenetic map locationch10q26.3. (See, FIG. 7).

SEQ ID No: 14 also includes the promoter region (bases 1.2839) for hpao.A typical GC-rich region resides just upstream from the 5′-end of theExon I. It contains nine consensus GC-boxes elements (2812 . . . 2799,2611 . . . 2598, 2632 . . . 2619, 2692 . . . 2679, 2712 . . . 2699, 2732. . . 2719, 2752 . . . 2739, 2782 . . . 2769, 2647 . . . 2634,), andanother GC-box element can be seen further upstream (1983 . . . 1996).Within this GC-rich region, there are motifs for cap signal fortranscription initiation (2877 . . . 2870, 2859 . . . 2866, 2842 . . .2849) and stimulating protein 1 (2812 . . . 2800, 2632 . . . 2620, 2692. . . 2680, 2712 . . . 2700, 2732 . . . 2720, 2752 . . . 2740, 2782 . .. 2770, 2647 . . . 2635, 2611 . . . 2595). Many other essential elementsare spread out in this promoter region: retroviral TATA box (649 . . .640), GATA-binding factor 3 sites (1803 . . . 1811, 987 . . . 995),GATA-binding factor 1 sites (986 . . . 995,17 . . . 26, 1802 . . . 1811,2264 . . . 2273, 1643 . . . 1652, 534 . . . 525, 1149 . . . 1158, 200 .. . 191), GATA-binding factor 2 sites (1643 . . . 1652, 986 . . . 995,1521 . . . 1512, 1613 . . . 1604), AP-4 (activator protein 4) bindingsites (1128 . . . 1119, 1119 . . . 1128, 1584 . . . 1593, 1778 . . .1787, 1865 . . . 1856, 282 . . . 273, 1602 . . . 1593), etc. Many othergene regulatory elements were also found in this promoter region: STREs(stress-response elements) (1692 . . . 1685, 2329 . . . 2322, 1037 . . .1044, 345 . . . 352, 426 . . . 433, 2612 . . . 2605, 719 . . . 712, 2814. . . 2807, 1717 . . . 1710), v-Myb (929 . . . 937, 246 . . . 1238),c-Myb (2109 . . . 2118, 2246 . . . 2255), tumor suppressor p53 (2090 . .. 2099, 578 . . . 569, 1566 . . . 1557). Thus, the human peroxisomal PAOgene is contemplated to be regulated by certain cellular conditions suchas stress and tissue development.

The mPAO cDNA sequence was used to BLAST search GenBank. The entire mpaosequence is contained in GenBank Accession No. NW_(—)000335, where ExonI starts at base No. 1069500 (gene transcript ID No. XM 133921.1). Thegene was found to map as 7 exons (See, FIG. 6 and SEQ ID NO:21). For SEQID NO:21, Exon I=bases 7477-7678, Exon II=8119-8609, Exon III=bases9301-9503, Exon IV=bases 10810-11066, Exon V=bases 13441-13554, ExonVI=bases 14172-14332, and Exon VII=bases 15805-16129. The mpao genemapped near the terminus of one arm of murine chromosome 7, atcytogenetic map location 7F4 (See, FIG. 7).

SEQ ID NO:21 also includes a promoter region (1 . . . 7476) upstreamfrom the 5′-end of Exon I. It contains a typical GC-rich region withseveral GC-boxes (7476 . . . 6801, 7383 . . . 7370, 7465 . . . 7452,6814 . . . 6801, 7441 . . . 7428), and a consensus motif required forpromoters. The consensus motifs for the cap signal (transcriptioninitiation) (7306 . . . 7313, 7393 . . . 7400, 7409 . . . 7416) andstimulating protein 1 (SP1) (7383 . . . 7371, 7465 . . . 7453) alsoreside in this region. Within this promoter region, other essentialelements are also present: CCAAT/enhancer binding protein beta (4162 . .. 4149, 1539 . . . 1526, 2673 . . . 2686), GATA-binding factor 1(3487 .. . 3500, 4046 . . . 4033, 576 . . . 589), GATA-binding factor 2 (3489 .. . 3498, 7009 . . . 7000, 4454 . . . 4445, 6289 . . . 6298, 4817 . . .4808), GATA-binding factor 3 (5451 . . . 5460, 5601 . . . 5592, 6041 . .. 6050, 4060 . . . 4051, 1439 . . . 1448, 5731 . . . 5740), cellular andviral TATA box elements (262 . . . 248, 2201 . . . 2215, 3587 . . .3573, 4426 . . . 4440). In addition, there are c-Myb (44 . . . 35, 190 .. . 199, 1481 . . . 1472, 872 . . . 881, 2386 . . . 2377, 3265 . . .3256), tumor suppressor p53 regulatory elements (622 . . . 612, 3331 . .. 332, 1789 . . . 1780, 1713 . . . 1722, 1521 . . . 1530) and STREs(3531 . . . 3538, 3494 . . . 3501, 2907 . . . 2900) motifs, which arecontemplated to control mpao expression under certain conditions.

EXAMPLE 3 Production and Purification of Recombinant mPAO and hPAO

In this example, the methods used to produce and purify recombinant mPAOand hPAO in bacteria are described. The pET29 c(+) vector (Novagen) wasused to construct a mpao prokaryotic expression system. E. coli DH5α wasused throughout for plasmid subcloning. First, a 5′-end fragment wasgenerated by PCR using mpao1 as the template for the gene-specificantisense primer mpao1R, and a sense primer containing Sac I and Nde Isites, and an ATG start codon 5′-GCGAGCTCAT ACATATGGCG TTCCCTGGCCCGCGG-3′ (SEQ ID NO:11), where the single underlines indicate the Sac Iand Nde I restriction enzyme sites, respectively. The PCR product wasrestriction digested, and the resulting Sac I/Bam HI fragment wassubcloned into pUC19-MPAO1 at Sac I and Bam HI sites to give pUC19-MPAO.Next, the full length cDNA of mpao was released by Nde I and Hind IIItreatment, and ligated into Nde I and Hind III sites of pET29c to give aplasmid denoted pET-MPAO. E. coli BL21 GOLD (DE3) (hvitrogen) wastransformed with this plasmid for mPAO production.

The E. coli transformant was grown on Luria-Bertani (LB) agar platesthat contained 30 μg/mL kanamycin. A single positive colony wasinoculated into 3 ml of LB broth containing 30 μg/mL of kanamycin(LB-kan) for overnight growth at 37° C. Approximately 500 μl of thisculture was then inoculated into 80 mL of fresh LB-kan medium and grownovernight. Five mL of the culture were transferred to each of five 2-Lflasks containing 1 L of fresh LB-kan medium, and the cultures grownovernight at 37° C. with shaking. Each flask was used to inoculate five12-L fermentors containing LB-kan media. Cell growth resumed at 30° C.with rapid stirring and vigorous aeration. When the cell density reachedan OD₆₀₀=0.6-0.7, IPTG was added to a final concentration of 50 μM.Bacterial growth was allowed to continue until the cell density reachedan OD₆₀₀=1.5-2.0. The cells were harvested by centrifugation. About 260g of cell paste were obtained from 60 L of growth media, and stored at−70° C.

Selected fractions for the various steps in the following purificationwere assayed for N¹-acetyl-Spm oxidase activity by a modification of apublished method (Holt et al., Anal. Biochem. 244:384-392 [1997]), whichmeasures the time-dependent formation of H₂O₂. The assay stock solutionswere: (A) 100 mM vanillic acid (in order to dissolve the acid, the pH ofthe solution was adjusted to 7 with KOH); (B) 50 mM 4-aminopyrine; (C)400 units/mL of horse radish peroxidase; (D) 50 mM N¹-acetyl-Spm; and(E) 100 mM glycine/KOH, pH 9.5, a pH near which the maximal activity wasreported to be attained (Hölttä, Methods Emzymol. 94:306-311 [1983]).Thirty μL each of solutions A through D were mixed with 2.86 mL ofsolution E, and 50 μL of this mixture was pipetted into individual wellsof a 96-well plate. Anywhere from 1 to 50 μL of a particular fractionwas added to a single well, and the relative activity of differentfractions was assessed visually by the time-dependent intensity changeof the developing pink color. The purity of various fractions was alsodetermined by SDS-PAGE using pre-cast, 10-20% Tris-HCl Ready Gels(Bio-Rad), following the manufacturer's instructions.

The purification was initiated by placing 260 g of the frozen E. colicell paste in a large beaker with 10 mM MOPS buffer, pH 7.25. The pH ofthe MOPS buffer was adjusted at 21° C. to yield an estimated pH of 7.35at 4° C. (e.g., the temperature at which the purification was carriedout unless noted otherwise). The total volume was approximately 800 mL.Once thawed, the cell paste was homogeneously suspended with a tissuegrinder with a large glass/teflon piston (Potter/Elvehjem), after whichthe mixture was passed twice through a Avestin Emulsiflex C5 Homogenizerat 15-20,000 psi. At this point, about 15 mg of solid FAD was dissolvedinto the solution. The supernatant was centrifuged at 50,000×g, for 30min and then dialyzed against 13 L of 10 mM MOPS buffer, pH 7.25 for 4hr, and against 13 L of fresh 10 mM MOPS buffer, pH 7.25 overnight. Thedialyzed solution was diluted to 2 L with the 10 mM MOPS buffer, pH7.25, and applied, with a flow rate of ˜20 mL/min, to a 14×25 cm DEAEcellulose (Whatman, DE-53) column that had been equilibrated with thisbuffer. The column was washed with 2 L of the same buffer, and then agradient elution from 0 to 400 mM KCl (8 L total gradient volume) in theMOPS buffer was initiated, at which time, the collection of 26-mLfractions was started. The flow rate was gradually increased from 20 to29 mL/min from the start to the finish of the gradient elution.Significant activity was spread widely from tubes 180 to 300 (4.7 to 7.8L), which were later combined. The volume was reduced to ˜500 mL usingAmicon pressure concentrators fitted with Amicon YM-10 membranes. Afterdissolving ˜15 mg of solid FAD, the resulting solution was dialyzed for4 hr against 13 L of 10 mM HEPES buffer, pH 7.8. The pH of the HEPESbuffer was adjusted at 21° C. to yield an estimated pH of 8.05 at 4° C.The solution was then dialyzed overnight against 13 L of fresh HEPESbuffer. The dialyzed sample was applied to a HEPES buffer-equilibrated5×39 cm DEAE Spherodex LS column packed with 100-300 μm sized beads(Sepracor/IBF). The column was washed with 500 mL of the 10 mM HEPESbuffer, before starting a 2.4 L gradient from 0 to 500 mM KCl, in thesame buffer. The column, with a 7 ft pressure-head, was run at themaximum flow rate, and once the gradient was started, 26 mL fractionswere collected. The majority of the activity eluted in tubes number82-108, which were combined (˜700 mL). The sample was concentrated to˜50 mL as described earlier, and then dialyzed for 4 hr, against 7 L of10 mM KH₂PO₄/KOH buffer, pH 7.2, and then, overnight, against 7 L offresh buffer.

The next step in the purification involved ion-exchange chromatographyon an MONO P HR 5/20 column (Amersham/Pharmacia) at room temperature,using the following solutions: (I) H₂O and (II) 1 M KH₂PO₄ mixed with 1M K₂HPO₄ to give 1 M potassium phosphate buffer, pH 7.2. After injecting2 mL of the sample at a flow rate of 1 mL/min, proteins were eluted withthe following gradient: 0% solution II at t=0; 0% to 1% II in 4 min; 1%to 30% II in 125 min. mPAO, which eluted from 38-41 min, was collectedas a single fraction and immediately placed on ice. This step wasrepeated until the entire sample had been processed. The mPAO fractionsfrom all the MONO P runs were combined, concentrated and washed into 1mM KH₂PO₄/KOH buffer, pH 7.2, using eight 2 mL Centricon-10concentrators (Amicon). After concentration, the final volume of themPAO containing solution was 2 mL in the 1 mM buffer.

The final step in the purification involved chromatography on a 1×10 cmceramic hydroxyapatite (HAP) column (e.g., type II HAP from Bio-Radpacked into an Amersham/Pharmacia HR 10/10 column) run at roomtemperature. The mPAO sample in 100 μL was diluted to 1 mL with H₂O, andthe entire sample injected immediately onto the HAP column with a flowrate of 2 mL/min. The elution was carried out as follows: from t=0 to 7min, 0% II; from t=7 to 9 min, 0 to 1% II; from t=9 to 19 min, hold at1% II. mPAO eluted as a broad peak from t=14 to 17 min. This step wasrepeated until the entire sample from the MONO P column had beenprocessed. The combined fractions were concentrated as for the MONO Pfraction and washed into 10 mM KH₂PO₄/KOH buffer, pH 7.2, to give a 3.68mg/mL mPAO solution, based on an ε₄₅₈=10,400 M⁻¹ cm⁻¹ and a MW=55,887 Dafor the enzyme. The enzyme was judged to be pure by SDS-PAGE, and byion-exchange chromatography on an analytical TSK DEAE 2SW column (0.4×25cm; a 0.75 mL/min flow rate, with a gradient from 1% to 50% solution IIin 30 min; a single sharp peak eluted at 23 min). The yield of pure mPAOwas 36.8 mg.

Using the conditions for the steady-state kinetic assay described below,mPAO was determined to be stable at 2-4 mg/mL when frozen at −20° C. or−80° C. and thawed through several cycles. However, at a concentrationof 30 μg/mL, activity was lost quickly after several freeze/thaw cycles,with more rapid loss occurring at −80° C. than at −20° C. When 33% (v/v)ethylene glycol was added, mPAO was stable for several cycles offreezing and thawing, for solutions containing 20 μg/mL to 4 mg/mL,regardless of the storage temperature. Thus, mPAO was stored at −20° C.in the presence of 33% (v/v) ethylene glycol. Ethylene glycolelimination and buffer exchange was accomplished easily by severalconcentration/dilution cycles using Centricon-10 centrifugeconcentrators.

Similarly, a full-length hPAO cDNA fragment was cloned into the pTrcHisAvector Invitrogen), and used to transform E. coli DH5α for production ofrecombinant hPAO. The recombinant hPAO contained a poly-histidine tag atits N-terminus for purification purposes. A centrifuged bacterial cellextract obtained from a 2 L culture, was applied to a Nickel-NTASuperflow column (Qiagen). The Nickel column preferentially bound theHis-tagged hPAO yielding several mg of pure hPAO. Recombinant His-taggedmPAO was also expressed to high levels with this system.

EXAMPLE 4 Spectral Characterization and Redox Properties of mPAO

In this example the methods used for the anaerobic reductive titrationof the purified recombinant MPAO are described. All UV-visible spectrawere recorded with a Hewlett-Packard 8452A diode arrayspectrophotometer. mPAO, in 50 mM KH₂PO₄/KOH buffer, pH 7.6, at 21° C.,was titrated anaerobically with a 0.541 μM sodium dithionite solution(See, FIG. 9). The dithionite solution was standardized by titratinganaerobically a FAD solution of known concentration (ε₄₄₅=11,300M⁻¹cm⁻¹). The anaerobic cuvette and other details of this procedure aredescribed elsewhere (Edmondson and Singer, J. Biol. Chem. 248:8144-8149[1973]; Efimov et al., Biochemistry 40:2155-2166 [2001]; and Engst etal., Biochemistry 38:16620-16628 [1999]). The anaerobic mPAO solutionalso contained 50 mM D-glucose, 3 μg of catalase, and 50 μg of glucoseoxidase to scavenge trace O₂. The spectral data was subjected to “FactorAnalysis” using the Spectrum SPECFIT program (Spectrum SoftwareAssociates). The increase in absorbance in the 380 nm region indicatesthe formation of the red radical, while the small increases in the 550to 700 nm region indicates the formation of a small amount of the blueradical (See, FIG. 9). The inset in FIG. 9, Panel B show a graph ofA₃₇₇, A₄₅₈ and A₅₉₀ versus the amount of DT added. From this plot, itwas determined that 15.2 mnol of DT were required to fully reduce theenzyme sample. These results provide valuable data regarding theinteraction of a portion of the FAD molecule and an alpha-helix of mPAO.The extinction coefficients at 458 nm and 273 nm were found to be 10,600and 99,200 M⁻¹ cm⁻¹, respectively. These values, when compared totheoretical values, confirm the presence of 1 molecule of FAD bound permolecule of PAO. The FAD was shown to be noncovalently bound bytreatment of a solution of mPAO with 5% trichloracetic acid. Thereleased flavin was quantitated by its fluorescence intensity relativeto that of a standard solution of FMN (Singer and McIntire, MethodsEnzymol. 106:369-378 [1984]).

The anaerobic titration of mPAO, in 50 mM KH₂PO₄/KOH buffer, pH 7.6, at21° C., with a solution of N¹-acetyl-Spm is contemplated. For thistitration, both the enzyme and substrate solutions contain D-glucose,catalase and glucose oxidase.

EXAMPLE 5 Steady-State Kinetic Analysis of mPAO

In this example the methods used to examine mPAO steady state kineticsare described. Spectrophotometric assays were done at 30° C. in apotassium phosphate buffer (e.g., 50 mM KH₂PO₄/KOH buffer, pH 7.6saturated in air or in pure oxygen), using a published procedure whichprovides a continuous monitor of the H₂O₂ produced (Holt et al., Anal.Biochem. 244:384-392 [1997]). These assays were done in 1 mL cuvettewith 0.8 mL of solution containing varying amounts of substrate, 1 mMvanillic acid, 0.5 mM 4-aminopyrine, 4 units of horseradish peroxidase,and 0.1-0.2 μg of mPAO. The reactions were monitored at 498 nm with aUVIKON 840 spectrophotometer (KONTRON Instruments) for the formation ofthe quinoneimine dye (ε₄₉₈=4,650 M⁻¹cm⁻¹ at pH 7.6), the condensationproduct of vanillic acid and oxidized 4-aminopyrine. The latter isproduced from 4-aminopyrine by the action of horseradish peroxidase thathas been oxidized by H₂O₂ (Holt et al., supra [1997]). The assays weredone at 25° C. by varying the concentration of the amine substrate,while the oxygen concentration in the assay solution was constant at theair-saturating level of 237 μM. The data were fit, by nonlinearregression to steady-state kinetic equations (McIntire et al., Biochem.J. 228:325-335 [1985]), which provided the values, and standarddeviations thereof, for k_(cat)′, K_(S)′ and k_(cat)′/K_(S)′ (=Q, thespecificity constant). $\begin{matrix}{v = \frac{k_{cat}^{\prime}\lbrack S\rbrack}{{Ks}^{\prime} + \lbrack S\rbrack}} & {{Equation}\quad 1} \\{v = \frac{Q\lbrack S\rbrack}{1 + \frac{\lbrack S\rbrack}{{Ks}^{\prime}}}} & {{Equation}\quad 2}\end{matrix}$

It was determined that Spm, Spd, and Put (but not benzylamine), whichare either very poor or nonsubstrates, are inhibitors of the oxidationreaction of the best substrate, N¹-acetyl-Spm. The dissociationconstants, K_(D), for these and other amines were estimated in thefollowing manner. It was assumed that the competition was competitive,since in the presence of the inhibitors, at a saturating N¹-acetyl-Spmconcentration, the rate of the reaction was equal to the value of thetrue k_(cat). It was also assumed that the mechanism is of the ping-pongtype. Thus, the applicable steady-state equation is (Segal, EnzymeKinetics, John Wiley & Sons, New York, pp. 606-625 [1975]):$\begin{matrix}{v = {\frac{k_{cat}\lbrack S\rbrack}{{{Ks}\left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)} + {\lbrack S\rbrack\left( {1 + \frac{K_{0}}{\left\lbrack O_{2} \right\rbrack}} \right)}} = \frac{k_{cat}^{\prime}\lbrack S\rbrack}{{Ks}^{''} + \lbrack S\rbrack}}} & {{Equation}\quad 3}\end{matrix}$

On the left, k_(cat) and K_(S) are the steady-state parameters forN¹-acetyl-Spm (S), and I and K_(I) (=K_(D)), represent the inhibitor andits inhibition constant, respectively. K_(IO) and K_(O) are theinhibition constant and Michaelis constant for O₂, respectively. Theequation can be converted to the expression on the right side when:$\begin{matrix}{{{k_{cat}^{\prime} = \frac{k_{cat}}{\left( {1 + \frac{K_{0}}{\left\lbrack O_{2} \right\rbrack}} \right)}},{{Ks}^{''} = {{Ks}^{\prime}\left( {1 + \frac{\lbrack I\rbrack}{K_{I}}} \right)}},{{Ks}^{\prime} = \frac{Ks}{\left( {1 + \frac{K_{0}}{\left\lbrack O_{2} \right\rbrack}} \right)}}}\quad} & {{{Equations}\quad 4a},b,c}\end{matrix}$

The expressions for k_(cat)′ and K_(S)′ (at the fixed [O₂]=237 mM) arethe same as those when the inhibitor is absent. Since the values ofk_(cat)′ and K_(S)″ were predetermined, inhibition assays were carriedout where [S]≈K_(S)″, which, in the absence of I, would give a rate,ν≈0.5×k_(cat)″. In these assay, for each inhibitor, [I] was adjusted sothe apparent rate was somewhat less then 0.5 k_(cat)′. In thissituation, after substitution and rearrangement, Equation 1 reduces to:$\begin{matrix}{K_{I} = {\frac{\lbrack I\rbrack}{\frac{k_{cat}^{\prime}}{v^{\prime}}} - 2}} & {{Equation}\quad 5}\end{matrix}$

Since [I] and k_(cat)′ are known, and ν is the measured rate in thepresence of the inhibitor, the value of K_(I)′ can be estimated. It isassumed that these apparent K_(I)′ are good approximations of the trueK_(I) (=K_(D)) values. K_(I)′ values were estimated at four differentconcentrations of each of the following inhibitors, benzylamine, Spm,Spd, Put, and N⁸-acetyl-Spd. The calculated K_(I)′ values at the fourdifferent concentrations of each inhibitor were within ˜20% of eachother. The estimated K_(I)′ values provided in Table 1, are the averagesof these four values. If the reactions of the polyamine substrates andO₂ with mPAO obeys a ping-pong type mechanism, the apparent values,k_(cat)′/K_(S)′, are equal to the true k_(cat)/K_(S) values.

Some assays (oxygraph assays) were carried out by directly monitoring O₂consumption in air-saturated buffer ([O₂]=0.237 mM) or buffer saturatedwith pure O₂ at 30° C. ([O₂]=1.12 mM). The depletion of O₂ was detectedwith a Yellow Springs Instruments, Inc. Model 53 Oxygen Monitor equippedwith a Clark electrode. The true k_(cat) and K_(O) values for theoxidation of N¹-acetyl-Spm and N¹-acetyl-Spd were determined byprogress-curve analysis for reactions that were allowed to go tocompletion ([O₂]=0 at t=8). Following a published procedure (Goudar etal., Biochim. Biophys. Acta 1429:377-383 [1999]), the data were fittedto the integrated Mechaelis-Menton equation. The analyses were doneusing Maple VI (Windows 2000) software (Waterloo Maple, Inc.) running ona PC computer. Using the apparent K_(I)′ values (Table 1) as a guide,the saturating concentrations of N¹-acetyl-Spm and N¹-acetyl-Spd weremade high enough (3.7 mM) so that product inhibition by Spd and Put,respectively, was insignificant at all times during the reaction.Inhibition by the H₂O₂, formed as a product of polyamine oxidation bymPAO, was assessed by addition of 2 μL of a 30 mg/mL (30,000 units/mg)solution of catalase, after [O₂] reached zero. TABLE 1 Steady-StateKinetic Parameter for the Reaction of Various Amines (S) and O₂ (O) withPure mPAO k_(cat)′ K_(S)′ k_(cat)′/K_(S)′ K_(I)′ = K_(D) Compound (S)(sec⁻¹) (μM) (M⁻¹sec⁻¹) (μM) N¹-acetyl-Spm 4.53 ± 0.05 1.78 ± 0.1 (2.54± 0.01) × 10⁶ NA^(a) N¹-acetyl-Spd 4.85 ± 0.03 36.8 ± 1.1 (1.32 ± 0.03)× 10⁵ ND^(a) N⁸-acetyl-Spd 0 0 0 70 N¹,N¹²-diethyl-Spm 0.415 ± 0.012 150± 10 (2.77 ± 0.13) × 10³ ND N¹,N¹¹-diethyl-nor-Spm 1.93 ± 0.03 157 ± 7 (1.27 ± 0.03) × 10⁴ ND benzylamine 0 0 0 ∞ (infinity) Spm 0.175 ± 0.005716 ± 33 (2.47 ± 0.01) × 10² 750 Spd 0 0 0 190 Put 0 0 0 1,000 synthalin0 0 0 0.05 N-(3-aminopropyl) 1,10 0 0 0 35 decanediamine^(a)NA indicates not applicable, and ND indicates not determined.

TABLE 2 Steady-State Kinetic Parameter for the Reaction of VariousAmines (S) and O₂ (O) with Pure mPAO k_(cat) ^(a) K_(S)(calc)^(b)k_(cat)/K_(Q) ^(a) K_(Q) ^(a) Compound (S) (sec⁻¹) (μM) (M⁻¹sec−¹) (μM)N¹-acetyl-Spm 8.0 ± 0.8 3.1 ± 0.3 (4.4 ± 0.4) × 10⁴ 180 ± 20N¹-acetyl-Spd 13 ± 1  83 ± 8  (4.3 ± 0.4) × 10⁴ 301 ± 30^(a)The true k_(cat), K_(O), and k_(cat)/K_(O) values were determined byprogress-curve analysis of O₂ consumption, in the presence of asaturating concentration of N¹-acetylated polyamine substrate. Theconcentration of the substrate was assumed to be high enough to overcomeany inhibition by the polyamine product formed during the reaction. Thebuffer was saturated with pure O₂ (=1.12 mM). The errors were estimatedto be about 10%.^(b)The “true” K_(S) values calculated from the apparent valuesdetermined in air-saturated buffer ([O₂] = 0.237 mM]. The calculationswere done using the equation K_(S) = K_(S)′(1 + K_(O)/[O₂]) (Seeequations 4).

The catalase immediately converted each mole of H₂O₂ to 0.5 mol of O₂(since [O₂]=1.12 mM for pure O₂-saturated buffer, then [O₂]=0.56 mMimmediately after catalase addition). In the presence of catalase, O₂depletion should occur at 0.5× the rate as it does in the absence ofcatalase (unless H₂O₂ inhibition is operating). With catalase present,each molecule of O₂ will be converted eventually to H₂O (4-electronequivalents), rather than to H₂O₂ (2-electron equivalents). Thus, to getthe proper rates, all post-catalase Δ[O₂]/Δt values were multiplied by 2for the N¹-acetyl-Spm or N¹-acetyl-Spd case. It was found that eachresulting [O₂] versus time progress curve was superimposable on thecorresponding pre-catalase curve for [O₂]=0.56 to 0 mM. This indicatedthat H₂O₂ inhibition does not occur.

Thus, N¹-acetyl-Spm and N¹-acetyl-Spd were found to be good mPAOsubstrates and Spm was found to be a poor substrate. In addition,N¹,N¹²-diethyl-Spm (also known as N¹,N¹²-bis[ethyl]spermine or BE-Spm)and N¹,N¹¹-diethyl-nor-Spm (also known as N¹,N¹¹-bis[ethyl]norspermineor BEN-Spm), were also found to be very good substrates for mPAO. Thesepolyamines have been used widely to study the physiological effects ofpolyamine metabolizing enzymes. They down-regulate polyaminebiosynthetic enzymes, but dramatically up-regulate SSAT synthesis (Hu,and Pegg, supra [1997]; Kramer et al., supra [1999]). Thus, when thesesubstances are provided to cells, the intracellular levels ofN¹-acetyl-Spm and N¹-acetyl-Spd rise significantly due to SSAThyperproduction. This results in an increase in the extracellulartransport of the N¹-acetylated polyamines, as well as an increase in thelevel of cellular H₂O₂ and 3-acetamidopropanal, via their oxidation.Consequently, increased H₂O₂ levels induce an increase in SSATproduction (Seiler, Neurochem. Res. 25:471-490 [2000]). The increasedlevels of H₂O₂ (and possibly the increased amount of3-acetamidopropanal, which can be convert in the cell to cytotoxic,3-aminopropanal; Houen et al., supra [1994]) induces apoptosis. This canbe eliminated by treating the cells with the potent mechanism-based PAOinhibitor MDL 72527 (N¹,N⁴-bis(2,3-butadienyl)-butane-1,4-diamine).Induction of apoptosis by this mechanism maybe contributing to killingprecancerous cells, and contributing to the damage cause by ischemia andreperfusion (Mank-Seymour et al., supra [1998]; Lindsay and Wallace,supra [1999]; Chopra and Wallace, supra [1998]; Ha et al., Proc. Natl.Acad. Sci (USA) 94:11557-11562 [1997]; Ferioli et al., Biochem. Pharm.58:1907-1914 [1999]; Rao et al., J. Neurochem. 74:1106-1111 [2000];Hatcher et al., Soc. Neurosci. Abstr. Vol. 26:Program No. 769-9 [2000];and Zoli et al., Brain Res. Molec. Brain Res. 38:122-134 [1996];Ivanova, et al., supra [1998]; Ivanova, et al., supra [2002]).

Unless the levels of N¹-acetyl-Spm and N¹-acetyl-Spd are high enough tosuppress the oxidation of BEN-Spm or BE-Spm, their efficacy in inducingapoptosis may be diminished. This raises several considerations fordesigning anticancer drugs, drugs that can be used to minimize ischemicand reperfusion tissue damage, or drugs for developmental problemsdirected at altering polyamine metabolism. First, the efficacy of anypotential pharmaceutical may be diminished if it can be oxidizedefficiently by PAO. Alternatively, it is possible that the PAO-oxidizeddrug is the real therapeutic agent (e.g., that hyperinducing SSATproduction). Additionally, a PAO-oxidized drug may be toxic, or itstoxicity might be diminished by further PAO oxidation. Interestingly,BEN-Spm is currently in Phase II clinical trials (Bergeron et al., supra[2000]). However, an understanding of the mechanism(s) is not necessaryin order to make or use the present invention.

N-(3-Aminopropyl)-1,10-diaminodecane is known to be a ligand for thepolyamine domain of the N-methyl-D-aspartate (NMDA) receptor. Thisstrong antagonist interacts with both the NMDA and Gly recognition siteof the receptor (Yonada et al., Brain Res. 679:15-24 [1995]). As shownin Table 1, this polyamine analog is a good inhibitor of mPAO, with anapparent K_(I)′ of 35 μM. During the development of the presentinvention, it was found that this polyamine was not oxidized by mPAO,even though it has a terminal diaminopropyl group as does Spm. However,Spm, which has the same disposition of amino groups along its chain asN¹-acetyl-Spm, but lacks the terminal N-acetyl functionality, is a poormPAO substrate. Additionally, the spacing of the amino groups along thestraight chain of N-(3-aminopropyl)-1,10-diaminodecan is contemplated tolead to its improper alignment in the active site, for oxidation of theappropriate carbon center.

The spacing of amino groups suggests a reason why N⁸-acetyl-Spd is notoxidized by mPAO. It is suspected that this substrate, N¹-acetyl-Spm andN¹-acetyl-Spd all bind to mPAO with their N-acetyl group situated in thesame binding site. This is contemplated to help align substratescorrectly for oxidation. Thus, while the pertinent carbon center of theN-acetamidopropyl moieties (i.e., Ac-NH CH₂ CH₂*CH₂NH—] of N¹-acetyl-Spmand N¹-acetyl-Spd are properly position for oxidation by FAD, thiscarbon center is displaced by one methylene group in theN-acetamidobutyl moiety of N⁸-acetyl-Spd. As shown in FIG. 10, anon-oxidizable carbon center occupies the position favored by FAD (i.e.,Ac-NHCH₂CH₂*CH₂CH₂NH—).

Synthalin (1,10-bisguanidinodecane), with an apparent K_(I)′ of 50 nM,is an extremely potent inhibitor of mPAO. Previously, this substance wasthought to be only a potent potassium channel blocker and activator(Allard et al., FEBS Lett. 375:215-219 [1995]), and a noncompetitiveantagonist of the NMDA receptor (Reynolds, J. Pharmacol. Exp. Ther.263:632-638 [1992]; and Reynolds et al., J. Pharmacol. Exp. Ther.259:626-632 [1991]). That synthalin is a much better inhibitor of mPAOthan N-(3-aminopropyl)-1,10-diaminodecane is contemplated to be due tothe spacing of the cationic guanidino groups along the straight chainmolecule. For synthalin this spacing is contemplated to mimic thespacing of the terminal ammonium centers in N¹-acetyl-Spm andN¹-acetyl-Spd (See, FIG. 10). In fact, a terminal guanidino group ofsynthalin is contemplated to better imitate the acetamido group ofN¹-acetyl-Spm or N¹-acetyl-Spd, than does an amino group as present inN-(3-aminopropyl)-1,10-diaminodecane.

In addition, one or more derivatives of synthalin (See, FIG. 10) withdifferent number (n) of intervening methylene groups (n=10 forsynthalin) between the guanidino groups are contemplated to be efficientinhibitors of PAO. Also, synthalin or a derivative thereof with one orboth guanidino groups substituted with an amino group or an amidinogroup are contemplated to be effective PAO inhibitors. Furthermore,synthalin derviatives with alkylguanidino, alIylamidino, or alkylaminogroup(s) are contemplated to be effective inhibitors of PAO. Thus, someembodiments of the present invention provide synthalin variants definedby the following structure: R₁—(CH₂)_(n)—R₂, where both R₁ and R₂represent guanidino, amidino, amino, alkylguanidino, alkylamidino,alkylamino groups or any mixed pairing of these groups (e.g., forsynthalin, R₁ and R₁ are guanidino groups, and n equals 10 as shown inFIG. 10).

Amidino, amino, alkylguanidino, alkylamidino and alkyl amino groups areselected as suitable substitutes for the guanidino groups because, likethe guanidino group, they are all positively charged functionalities. Infact, this property is contemplated to be important for efficient PAOinhibition. In the above formula, “n” is variable because there isexpected to be an optimal methylene chain-length for effective PAOinhibition by different synthalin derivatives. Some of these derivativesare contemplated to possess high PAO inhibitory properties (e.g., asgood or better than synthalin), but have reduced interactions with othersystems. Synthalin derivatives with desirable features are identifiedusing the methods disclosed herein.

EXAMPLE 6 Analysis of PAO Oxidation Products

In this example, methods are described for determining the nature of theproducts of substrate oxidation by PAO. In a solution at pH 7.6containing catalase to destroy H₂O₂, mPAO and N¹-acetyl-Spm, at knownconcentrations, were allowed to react until the latter had beencompletely oxidized. mPAO and N¹-acetyl-Spd were similarly allowed toreact. Both reactions solutions contained a known amount ofN-(3-aminopropyl)-1,10-diaminodecane (Tocris Cookson) as an internalstandard. This standard was found not to be a PAO substrate. Aliquots ofthese reaction mixtures were treated with dansyl chloride(5-dimethylamino-1-naphthalenesulfonyl chloride) obtained from Sigma.The resulting dansylated polyamine products from each reaction wereanalyzed by reverse-phase high-pressure liquid chromatography (HPLC)following established procedures (Hunter, Methods in Molecular Biology:Polyamine Protocols, Morgan (ed.) Human Press, Totowa, N.J., Vol. 79,Chapter 14, pp 119-123). A Prodigy HPLC column (octadecylsilyl silicagel, 5 micron particle size, 0.46×5.0 cm; Phenomenex) was used, with aflow rate of 1 mL/min, and the following elution gradient: 0 to 45% Bfrom 0 to 0.1 min, 45 to 80% B 0.1 to 8 min, hold at 80% B from 8 to 11min, 80 to 90% B from 11 to 12 min. Detection was accomplished with aGilson Spetra/Glo fluorescence detector using a 7-51× excitation filter(330-400 nm) and a 3-72M emission filter (460-600 n m). Quantificationand identification of the peaks was accomplished by dansyl chloridetreatment and HPLC analysis of a standard solution containing knownamounts of Spm, Spd, putrescine, N¹-acetyl-Spm, N¹-acetyl-Spd, andN-(3-aminopropyl)-1,10-diaminodecane (internal standard). The completePAO oxidation of N¹-acetyl-Spm and N¹-acetyl-Spd was found to producestoichiometric amounts of Spd and putrescine, respectively. Thisconfirms that the PAO described herein is the classical mammalianperoxisomal N¹-acetyl-polyamine oxidase (Höltta, Biochemistry 16:91-100[1977]; Bolkenius and Seiler, Int. J. Biochem. 13:287-292 [1981];Höltta, Methods Enzymol. 94:306-311 [1983]; Seiler, Prog. Brain Res.106:333-344 [1995]).

Aliquots of each of the above described enzyme-reaction solutions weretreated with an equal volume of 2,4-dinitrophenylhydrazine (AcrosOrganics) solution (100 mg in 94 mL ethanol+6 mL of concentrated HCl). Astandard solution containing known amounts of acrolein (Acros Organics),3-acetamidcpropanal and 3-aminopropanal (infra) was treated in anidentical manner. This treatment converts the three aldehydes to thecorresponding 2,3-dinitronitrophenylhydrzone derivatives. Theenzyme-reaction and standard solutions were analyzed by reverse-phaseHPLC: Prodigy octadecylsilyl silica gel hplc column (supra): flow rate,1 mL/min; gradient elution-0% B for 0.5 min, 0 to 35% B from 0.5 to 1.5min, hold at 35% B from 1.5 to 5.0 min, 35 to 100% B from 5.0 to 9.0 min(solutions A and B were H₂O and acetonitrile, respectively, bothcontaining 0.5% (v/v) trifluoroacetic acid). The HPLC system usedSpetraSYSTEM P2000 gradient pumps, a UV6000LP Diode Array Detector, andthe ThermoQuest ChromQuest Chromatography Data System (Thermo SeparationProducts). The 368-nm chromatograms were used for quantitative analyses.The complete oxidation of either N¹-acetyl-Spm or N¹-acetyl-Spd by PAOwas found to produce an equivalent amount of 3-acetamidopropanal. Thisresult further substantiates the conclusion that PAO described herein isthe classical mammalian peroxisomal N¹-acetyl-polyamine oxidase (Höltta,Biochemistry 16:91-100 [1977]; Bolkenius and Seiler, Int. J. Biochem.13:287-292 [1981]; Höltta, Methods Enzymol. 94:306-311 [1983]; Seiler,Prog. Brain Res. 106:333-344 [1995]).

3-Acetamidopropanal and 3-aminopropanal were synthesized using a methodfamiliar to those skilled in the art of organic synthesis. A solution of1-amino-3,3-diethoxypropane (the diethyl acetal of 3-aminopropanal)(Acros Organics) in dry pyridine was reacted with acetic anhydride toproduce 1-acetamido-3,3-diethoxypropane (the diethyl acetal of3-acetamidopropanal). This compound has not been previously described.After removing most of the pyridine and acetic anhydride by rotaryevaporation at high vacuum, the resulting liquid was purified bydistilling at high vacuum: bp 111° C. (0.45 mm Hg).1-Acetamido-3,3-diethoxypropane and 1-amino-3,3-diethoxypropane werehydrolyzed by mixing 1 volume of either with 10 volumes of 1.5 N HCl.Within 1 min, these diethyl acetals were converted to3-acetamidopropanal and 3-aminopropanal, respectively, which were usedimmediately for the analysis described in the previous paragraph.

EXAMPLE 7 Analysis of mPAO and hPAO Expression

In this Example, methods used to determine the tissue specificity ofmPAO and hPAO transcription are described. The “Rapid Scan GeneExpression Panel” (Origin) was used to examine mPAO expression invarious mouse tissues and developmental stages. Briefly, total RNA fromeach sample was subjected to oligo-dT selection, and the first strandcDNAs were generated from poly-A+ mRNA using oligo-dT primers andMoloney murine leukemia virus (MMLV) reverse transcriptase. A 540 bpfragment of mpao was PCR amplified from mouse cDNA samples using twogene specific primers: sense primer, 5′-TCGGAAGAGA ACCAGCTTGT GG-3′ (SEQID NO:12); and antisense primer 5′-CAATGACATG ATGTGCAGGC A-3′ (SEQ IDNO:13). As a control, a 570 bp portion of the β-actin gene was alsoPCR-amplified, using primers provided by the manufacturer. The 24 mousecDNA samples were serially-diluted over a 4-log range and arrayed into a96-well PCR plate. The PCR reaction was carried out as follows after a 3min hot start at 94° C: 35 cycles of 94° C. for 30 sec, 55° C. for 1 minand 72° C. for 2 min. The amplified fragments were electrophoresed on anagarose gel and the fluorescence intensity of the ethidiumbromide-stained bands was used to measure relative expression levels ofβ-actin in the upper panel and murine PAO in the lower panel of FIG. 11.In this figure, M denotes the lane containing the DNA standards, and thenumbered lanes represent mRNA from the following tissues: 1) brain; 2)heart; 3) kidney; 4) spleen; 5) thymus; 6) liver; 7) stomach; 8) smallintestine; 9) muscle; 10) lung; 11) testis; 12) skin; 13) adrenal gland;14) ovary; 15) uterus; 16) prostate gland; 17) 8.5 day old embryo; 18)9.5 day old embryo; 19) 12.5 day old embryo; 20) 19 day old embryo; 21)virgin breast; 22) pregnant breast; 23) lactating breast; and 24)involuting breast. Embryo ages are given in days post-conception. Thelow levels of β-actin DNA in lanes 1, 10, 11 and 12 are due to asupplier's error. As shown in FIG. 11, the relative mPAO expressionlevels were found to be: liver>adrenal gland≈ovary≈pregnantbreast>spleen≈lactating breast>19 day old embryo>heart≈12.5 day oldembryo>uterus>9.5 day old embryo>stomach≈small intestine≈involutingbreast>thymus≈muscle≈lung>prostate (barely detectable). The levels ofmPAO in lanes 10-12 (e.g., lung, testis, and skin), could not beestimated due to the low levels of mRNA in these samples.

Additionally a multiple tissue northern (MTN) blot was probed with a³²P-labeled fragment of human PAO to determine the distribution of PAOmRNA in human tissues. The MTN blot (Clontech) contained approximately 2μg of polyA+ RNA per lane from 16 different human tissues. The hPAOprobe was generated from the human EST containing plasmid (GenBank)after Hind III and Eco RI digestions. The hPAO fragment was labeled with³²P-dATP via a random-primer labeling method. Hybridization was carriedout at 65° C. for 3 brs using the ExpressHyb hybridization solution(Clontech) per the manufacturer's instructions. After hybridization theblot membranes were exposed on X-ray film. As shown in FIG. 12, therelative hPAO expression levels were found to be:testis>>>>liver>heart≈skeletalmuscle≈pancreas≈kidney>spleen≈prostate>ovary≈small intestine≈peripheralblood leukocytes≈brain>placenta>lung≈colon≈thymus.

EXAMPLE 8 Analysis of hpao Expression in Cancer Cells

In this example, the sequencing of hpao and expression of hpao mRNA fromhuman tumor cells is described. By screening GenBank using the hpaosequence as the probe, an altered form of hpao mRNA was found (GenBankAccession No. AW662266) in a sequence derived from a genitourinary tracthigh-grade transitional cell tumor (TCC). Upon completely sequencing theinsert of this cDNA clone, a 10-bp deletion was observed at the splicejunction of Exons III and IV (See, FIG. 6, Pane D). This deletion ispredicted to result in early termination of hpao mRNA translation and toproduce an inactive, truncated form of hPAO. A short repeat (CTTAGG)occurs within a 16-bp fragment 5′-CTTAGGTTTT CTTAGG-3′ (SEQ ID NO:15) inthe spliced mRNA of full-length hpao. While the short repeat sequencesuggests that improper splicing had occurred to produce the 10-bpdeletion 5′-TTTTCTTAGG-3′ (SEQ ID NO:16). It is not known whether thisdeletion occurred within the genome or whether it occurred duringtranscription in the TCC cells. Additionally, a search of the human ESTbank uncovered an EST clone from a Soares ovary tumor, that lacks V(GenBank Accession No. AA293017), and a colon cancer-derived EST thatlacks Exon IV (GenBank Accession No. AW973180). In contrast, hpao cDNAisolated and sequenced from normal human liver, placenta and testis,were found to be complete. It is therefore contemplated that alterationsin hpao may contribute to the initiation and/or progression of somecancers.

Interestingly, an hpao sequence derived from a fetal brain tissue clone(GenBank Accession No. BI91922), which contains an insert extending fromhpao Exon I to Exon VI, is missing the 3′-end of Exon II and all of ExonIV. Thus, altered transcription of hpao mRNA is contemplated to occur infetal tissue.

In addition, a reverse-transcriptase-PCR (RT-PCR) experiment wasperformed to analyze the expression of hpao in OVCAR-3 and HL-60 cells.Briefly, about 10⁶ to 10⁷ cells were harvested and the total RNA of eachsample was isolated by using the RNeasy miniprep kit (QIAGEN). A 1 μgmRNA sample was used for cDNA synthesis with the MMLV reversetranscriptase following the manufacturer's instructions provided withthe Advantage RT-for-PCR Kit (Clontech). Human liver and placenta polyAmRNA were also obtained from Clontech. cDNA amplification was achievedfollowing the SMART RACE™ cDNA Amplification method (Clontech). A pairof gene specific primers was generated based on the hpao cDNA sequence;hpao1 was the sense primer 5′-AGGCAGCCTT TCCCGGGGAG ACCTTTC-3′ (SEQ IDNO:17), and hpao2R was the antisense primer 5′-TCTCCATGAA CTCAGACTCAAGT-3′ (SEQ ID NO:18). Positive control amplimers for human G3PDH(glyceraldehyde-3-phosphate-dehydrogenase) were also used; the senseprimer was 5′-TCCACCACCC TGTTGCTGTA G-3′ (SEQ ID NO:19) and theantisense primer was 5′-GACCACAGTC CATGCCATCA CT-3′ (SEQ ID NO:20). PCRwas performed at 94° C. for 3 min, 25 cycles at 94° C. for 10 sec, 68°C. for 1 min, and 72° C. for 1 min.

The results of the RT-PCR experiment indicated that the levels of hpaomRNA were very low in both OVCAR-3 and HL-60 carcinoma cells incomparison with that observed in human liver and placenta cells (See,FIG. 13). The decrease in hpao mRNA is contemplated to be due to downregulation of hpao transcription in tumor cells. Moreover, since tumorsuppressor genes are frequently down regulated in cancer cells, it iscontemplated that hpao functions as a tumor suppressor gene.

EXAMPLE 9 Analysis of hpao Expression During Apoptosis

In this Example, the method used to examine hpao mRNA production bycultured cells undergoing apoptosis is described. Briefly, cell deathwas induced by adding N¹-acetyl-Spm to the growth medium of OVCAR-3human ovarian carcinoma cells, and HL-60 human promyelogenous leukemiacells that had undergone 48 hrs of growth. Approximately 50 μMN¹-acetyl-Spm killed 100% of the OVCAR-3 cells within 20 hrs. HL-60cells, in contrast, survived for 24 hrs at all tested N¹-acetyl-Spmconcentrations. In addition, although 100% of the HL-60 cells survived a72 hrs exposure to 0.05 mM N¹-acetyl-Spm, 30%, 50% and 80% of thesecells were killed in the presence of 0.1 mM, 0.2 mM, and 0.5 mMN¹-acetyl-Spm, respectively (See, FIG. 14). Clearly both OVCAR-3 andHL-60 cells can be induced to undergo apoptosis by N¹-acetyl-Spm, butwith different susceptibilities. The results of areverse-transcriptase-PCR experiment indicated that significant levelsof hpao mRNA were induced in OVCAR-3 cells very quickly after exposureto N¹-acetyl-Spm. There was no change in the level of hpao mRNA levelsin the HL-60 cell line during a 48 hr exposure to high concentrations ofN¹-acetyl-Spm, which correlated with a decreased susceptibility of thesecells to N¹-acetyl-Spm-induced apoptosis.

EXAMPLE 10 Structure of mPAO

In this Example, the analysis of the structure of mPAO was initiatedwith an alignment of several known flavoprotein amine oxidase amino acidsequences. The alignment, which was accomplished using the CLUSTALW(version 1.8) server found at the European Bioinformatics Institutewebsite, was refined further manually. The manual aligning and alignmentdisplay was done using the Windows program GeneDoc (version 2.6.002)available at the Pittsburgh Supercomputing Center Biomedical Initiativewebsite. The sequences used to produce this alignment included:peroxisomal bovine PAO (bPAO), peroxisomal human PAO (hPAO), peroxisomalmurine PAO (mPAO), cytosolic human Spm oxidase (GenBank Accession No.AY033889; NCBI Accession No. BAA91360; Wang, et al., supra [2001];Vujcic, et al., supra [2002]), cytosolic murine Spm oxidase (GenBankAccession No. BC004831; NCBI Accession No. AAH0483 1; Vujcic, et al.,supra [2002]), human MAO-A (hMAO-A; GenBank Accession No. M69226), humanMAO-B (hMAO-B; GenBank Accession No. M69177), Salmo gairdneri (fish) MAO(fMAO; GenBank Accession No. L37878), Mycobacterium tuberculosis amineoxidase (Mt-AmOx; GenBank Accession No. AL021646), Micrococcus rubensputrescine oxidase (Put-Ox; GenBank Accession No. D1251 1), Zea mays(corn) PAO (cPAO; GenBank Accession No. AJ002204), Micro luteus tyramineoxidase (Ml-TyrOx; GenBank Accession No. 3298360), Aspergillus niger MAO(MAO-N; GenBank Accession No. L38858), and Candida boidiniiN¹-acetyl-Spd oxidase (CB_N-SpdOx; GenBank Accession No. AB018223). Onlythe cPAO sequence has a recognizable N-terminal transport signalsequence. In contrast, a tripeptide peroxisomal transport signal ispresent at the C-termini of bPAO, hPAO, mPAO, MAO-N and Cb_N-SpdOx;peroxisomal transport consensussequence=-[S/A/C/P]-[K/H/R]-[I/L/M](Gould, et al., J. Cell Biol.108:1657-1664 [1989]). Extended C-terminal regions of hMAO-A, hMAO-B andfMAO are thought to be anchors that hold these proteins to the outersurface of mitochondria (Binda, et al., Nature Struct. Biol. 9:2-26[2002]).

From the alignment, 2 highly conserved regions were identified: one nearthe N-termini of these enzymes is a clearly identifiable β-α-β consensusdomain, which interacts with the ADP moiety of FAD (Schilling and Lerch,Biochim. Biophys. Acta 1243:529-537 [1995]; and Wierenga, Terpstra, andHol, J. Mol. Biol. 187:101-107 [1986]); the other located at theC-termini is also involved in FAD binding. Moreover, located close tothe C-termini is a moderately conserved region that harbors the Cysresidues of the monoamine oxidases that are covalenily linked to FAD:Cys⁴⁰⁶ (hMAO-A) and Cys³⁹⁷ (hMAO-B). In bPAO and mPAO, a Ser (Ser⁴²⁹ ofmPAO) aligns with these Cys residues. The CLUSTALW analysis provided thefollowing percent identities (percent similarities) between mPAO andother flavin-containing amine oxidases: hPAO, 79% (89%); bPAO, 73%(82%); cytosolic human Spm oxidase, 36% (53%); cytosolic murine Spmoxidase, 36% (53%); Micrococcus rubens Put-Ox, 19% (32%); cPAO 19%(34%); Salmo gairdneri MAO, 17% (30%); human MAO-B, 16% (30%);Mycobacterium tuberculosis amine oxidase, 16% (30%); Candida boidiniiN¹-acetyl-Spd oxidase, 16% (32%); human MAO-A, 15% (30%); Micro luteustyramine oxidase, 13% (28%); Aspergillus niger MAO-N, 12% (25%).Overall, the amino acid sequence identity between mPAO and the otherflavoprotein amine oxidases is rather low (e.g., generally less than20%, except for the 36% identity to the newly discovered human andmurine Spm oxidases). Thus, it is contemplated that the bovine, murineand human peroxisomal PAOs described herein, represent a new subclass ofmammalian amine oxidases.

Even so, there appears to be a great deal of conservation of the basicstructural elements of mPAO and cPAO. For example for mPAO, thepositive-ends of the α-helical dipoles that interact with thediphosphates (the sequence from residues 14-26) and the N1/C2 O locus ofFAD (the sequence from residues 475-491) are conserved, as are someelements of the Rossmann fold (the sequences from residues 6-38corresponding to the βαβ motif, 261-282, 291-305, 420-431, and 476-494)(Dym and Eisenberg, Protein Sci 10:1712-1728 [2001]). Three conservedregions that are close to and possibly interact with FAD and substrate,are those defined by residues 38-50, 56-64, and 216-243. Two otherconserved regions that are remote from FAD and the substrate-bindingsite are located at residues 315-325 and 371-376. Therefore, thestructure of mPAO was modeled using the known cPAO structures (Binda etal., Structure 7:265-276 [1999]) as templates. The coordinates of the1.9 angstrom X-ray structure for corn PAO (cPAO) and the cPAO/MDL75257complex are available in PDB files 1B37 and 1B5Q respectively (Binda etal., supra [1999]). Initially the PC program Swiss-PdbViewer version3.5b3, found on the ExPASy Molecular Biology Server website, displaysthe three-dimensional structures of cPAO/MDL template and a linearα-helix structure of the target protein mPAO. Using the “Magic Fit”option, the program threads the mPAO sequences onto the cPAO structure.The program displays the superimposed structures of the two oxidases andthen the second template cPAO, is superimposed onto these structures.The sequence alignment of all three proteins is shown on the computermonitor. The fit of the mPAO sequence to the cPAO/MDL structure wasrefined further by using the “Iterative Magic Fit” option. Visualadjustments were made to the sequence alignments, resulting in changesto the predicted crude mPAO structure (e.g., gaps in the crude mPAOstructure are spanned by unusually long bonds). Multiple “good” crudemPAO structures were generated in this way, each having a differentalignment with the templates. The coordinates of each crude mPAOstructure and the corresponding structural information for thetemplates, cPAO/MDL and cPAO, in PDB format, were sent to SWISS-MODELlocated at the ExPASy Molecular Biology Server web site. With this site,molecular mechanic energy minimizations of the crude mPAO structureswere carried out (Optimized Mode) using the GROMOS96 force fieldprogram. The program failed to provide valid structures for about 30-40%of the crude models (i.e., GROMOS96 program crashed). For the remainingmodels, PDB coordinate files with “refined” mPAO structures wereobtained. Each file also contained the structures for cPAO and cPAO/MDL.When any file was viewed with the Swiss-PDB Viewer, all three structureswere superimposed. Those portions of the modeled mPAO structure thatwere ill-defined or that produced unfavorable interactions weredisplayed in red. The inspection of twelve mPAO structures, indicatedthat, while the overall tertiary structures differed, thestereochemistry of the substrate and FAD binding sites were remarkablywell conserved. One structure with the fewest ill-defined regions andunfavorable interactions was selected. The PDB file for this structurewas edited with a word processor to remove all the coordinates for thecPAO and the cPAO/MDL structures except for the coordinates for the FADof cPAO, yielding a PDB file with the coordinates for the “refined” mPAOstructure bound to FAD.

The mPAO amino acid sequence was sent to the “Predict Protein” web siteof the European Molecular Biology Laboratory, and the “Psi-Pred” website of the University College London, which returned predictedsecondary structures for mPAO. These secondary structures were comparedwith that for the Swiss Model 3-D structure of mPAO/MDL. As shown inFIG. 15, the secondary structure for Psi-Pred shows a secondarystructure very similar to that derived from the modeled mPAO structure.

The mPAO structure was refined further by performing a molecularmechanics energy minimization using the CHARMM22 program running on aDEC Alpha computer, resulting in some minor changes. Next the MDL 27527structure from cPAO was placed into the original GROMOS96 mPAOstructure, in order to CHARMM-minimize the energy of the structure. Thesubstrate, N¹-acetyl-Spm was placed into the active site of mPAO and theenergy of this complex was again minimized with CHARMM. The ribbonstructures of the mPAO/MDL 72527 complex is shown in FIG. 16, and theribbon structures of the mPAO/N¹-acetyl-Spm complex is shown in FIG. 17.A large number of acidic amino acyl residues are in the vicinity of thesurface entrance to substrate binding channel : Glu⁵², Glu⁸⁴, GlU⁸⁵,Asp⁹⁰, Asp²⁰⁶, Asp²⁸⁵, Glu³¹⁴, Glu²¹⁶, Glu³¹⁷, Glu³²¹, Asp³²³, Glu³³²,Asp³³³, Asp³³⁹, Glu³⁶², and GlU³⁸⁰.

As with the cPAO x-ray structure (Binda, et al., supra [1999]), the mPAOmodeled structure is composed of two distinct domains, asubstrate-binding domain and a flavin-binding domain. Thesubstrate-binding domain is composed of the following segments of themodeled mPAO structure: Ser88-Cys¹⁸⁵ and Gly³⁰⁹-Arg⁴¹⁹. Theflavin-binding domain consists of mPAO segments: Met¹-Leu⁸⁷,Cys¹⁸⁶-Leu³⁰⁸ and Trp⁴²⁰-Leu⁴⁹⁹ (FIG. 5 and FIG. 17). At the interfaceof these domains, there are numerous amino acyl residues that areinvolved in substrate and inhibitor binding (supra). Much of theFAD-binding domain of mPAO is defined by a classical Rossmann fold (Dymand Eisenberg, supra [2001]), which interacts primarily with ribityl-ADPmoiety of the flavin (top part of the protein structure in FIG. 16)(supra).

A continuation of these modeling studies is contemplated. In particular,the anticonvulsant, milacemide is placed in the active site of mPAO andthe energy of this complex is then minimized with CHARMM force fieldparameters. Similarly, the substrate MDL 27659 is placed in the activesite of mPAO for energy minimization. Further refinements are carriedout by energy minimizing molecular mechanics calculations of the mPAOand mPAO/X complex structures in a water box. Now that the structure ofMAO-B is known (Binda et al., Nature Struct Biol 9:22-26 [2002]), itwill be possible to use simultaneously the structures of cPAO and MAO-Bas templates to model the structures of mPAO and hPAO.

Since the structures of the X component (e.g., N¹-acetyl-Spm, MDL 72527,milacemide, and MDL 27695) of the complex vary considerably, thesestudies provide an idea of the flexibility, size, and topology of theactive site of the PAO/X complexes. The stereochemistry in the vicinityof the active site of these complexes also indicates those groups thatare essential for binding and oxidation of any of these secondary aminesubstrates. This information is invaluable for designing drugs that aremore or less specific to PAO. As the substrate-binding pocket in thevicinity of FAD is quite large, the addition of a side group to thesubstrate or inhibitor is contemplated to increase or decreasespecificity as needed.

The active site structures of modeled mPAO and the X-ray structure ofcPAO have been compared. All residues within a 15 angstrom sphere fromthe N5-position of FAD of the superimposed isoalloxazine rings wereviewed. There are significant differences, as well as similarities. Thisfinding is not unexpected given that the natural substrates for mPAO andcPAO are different. The substrates for mPAO are N¹-acetyl-Spm andN¹-acetyl-Spd. In contrast, the substrates for cPAO are Spm and Spd,which are poor substrates for mPAO. Additionally, the carbon centersoxidized by mPAO and cPAO differ.

The veracity of the “best” mPAO structure was tested by sending its PDBcoordinate to the PROCHECK web site of the University College London(Laskowski et al., J. Appl. Cryst. 26:283-291 [1993]). For the PROCHECKanalysis the “resolution” was automatically set to 2.0 angstroms. ThePROCHECK program “checks the bond lengths, bond angles, peptide andside-chain ring planarities, chirality, main-chain and side-chaintorsion angles, and clashes between nonbonding pairs of atoms”(Marti-Renom et al., Ann. Rev. Biophys. Biomol. Struct. 29:283-291[2000]). The results of this analysis indicated that the structure hadvery few bad or unusual structural features. For example, a Ramachandrananalysis indicated that 83.5% of the residues are in the most favoredregion, 13.7% are in the allowed regions, 1.4% in generously allowedregions and 1.4% in the disallowed regions. Also the structure did notshow any aromatic ring or peptide bond distortions and there were only afew residues with “distorted” bond lengths or bond angles.

In summary, the basic features of the stereochemistry of the active siteare well-represented by the structures determined in this Example. Thisconclusion is based upon the finding that when the substrate israndomly-positioned within its binding site in mPAO, the carbon atomsfrom which a hydrogen is removed aligned optimally with the N5-positionof the flavin isoalloxazine ring. Apparently, the residues Glu⁸⁴ andASP³³⁹ are crucial for providing a binding register by interacting withthe amino groups of the substrate.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention, which are obvious to those skilled inrelevant fields, are intended to be within the scope of the presentinvention.

1. An isolated nucleic acid that comprises a sequence selected from thegroup consisting of a gene encoding a peroxisomal acetylpolyamineoxidase protein of a mammal, a gene encoding a biologically activeportion of said peroxisomal acetylpolyamine oxidase protein, and a geneencoding a biologically active variant of said peroxisomalacetylpolyamine oxidase protein.
 2. The isolated nucleic acid of claim1, wherein said mammal is selected from the group consisting of a cow, amouse and a human.
 3. The isolated nucleic acid of claim 2, wherein saidnucleic acid is selected from the group consisting of the open readingframes of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID No:5.
 4. The isolatednucleic acid of claim 2, wherein said nucleic acid encodes a proteinselected from the group consisting SEQ ID NO:2, SEQ ID NO:4, and SEQ IDNO:6.
 5. The isolated nucleic acid of claim 1, wherein said nucleic acidencodes a protein with amine oxidizing activity.
 6. The isolated nucleicacid of claim 5, wherein said substrate for said amine oxidizingactivity is selected from the group consisting of N¹-acetyl-Spm,N¹-acetyl-Spd, N¹, N¹²-diethyl-Spm, N¹, N¹¹-diethyl-nor-Spm, and Spm. 7.A vector comprising the nucleic acid of claim
 1. 8. The vector of claim7, further comprising a promoter operatively linked to said nucleicacid.
 9. A host cell comprising the vector of claim
 7. 10. The host cellof claim 9, wherein said host cell is located in an animal.
 11. A hostcell comprising a disruption of the gene of claim
 1. 12. The host cellof claim 11, wherein said host cell is located in an animal.
 13. Anisolated mammalian nucleic acid sequence selected from the groupconsisting of the open reading frames of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5 and sequences that hybridize to the complement of the openreading frames of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, underconditions of low stringency, and wherein said isolated nucleic acidsequence encodes a polypeptide having amine oxidizing activity.
 14. Acomposition comprising an isolated protein selected from the groupconsisting of a peroxisomal acetylpolyamine oxidase of a mammal, abiologically active portion thereof, or a biologically active variantthereof.
 15. The composition of claim 14, wherein said mammal isselected from the group consisting of a cow, a mouse and a human. 16.The composition of claim 15, wherein said peroxisomal acetylpolyamineoxidase comprises a sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
 17. The composition of claim 14,wherein said peroxisomal acetylpolyamine oxidase has amine oxidizingactivity.
 18. The composition of claim 17, wherein the substrate forsaid amine oxidizing activity is selected from the group consisting ofN¹-acetyl-Spm, N¹-acetyl-Spd, N¹, N¹²-diethyl-Spm,N¹,N¹¹-diethyl-nor-Spm, and Spm.
 19. The composition of claim 14,wherein said peroxisomal acetylpolyamine oxidase is a recombinantperoxisomal acetylpolyamine oxidase protein.
 20. The composition ofclaim 19, wherein said peroxisomal acetylpolyamine oxidase proteinfurther comprises an affinity tag.
 21. A method for detecting mammalianperoxisomal acetylpolyamine oxidase expression in a cell comprising thesteps of: a) providing: i) a sample from a mammalian subject, and ii) atleast one reagent capable of specifically detecting mammalianperoxisomal acetylpolyamine oxidase expression; and b) contacting saidsample with said at least one reagent under conditions suitable forbinding said at least one reagent to a mammalian peroxisomalacetylpolyamine oxidase gene product.
 22. The method of claim 21,wherein said mammalian peroxisomal acetylpolyamine oxidase gene productcomprises mRNA, and wherein said at least one reagent comprises anucleic acid probe of at least 12 nucleotides in length thatspecifically hybridizes under conditions of high stringency to said mRNAor to cDNA corresponding to said mRNA.
 23. The method of claim 22,wherein said contacting is accomplished by a technique selected from thegroup consisting of polymerase chain reaction and Northern blotting. 24.The method of claim 21, wherein said mammalian peroxisomalacetylpolyamine oxidase gene product comprises protein, and wherein saidat least one reagent comprises an antibody that binds to said protein.25. The method of claim 24, wherein said contacting is accomplished by atechnique selected from the group consisting of enzyme-linkedimmunosorbent assay, Western blotting, immunofluorescence analysis,immunohistochemistry and flow cytometry.
 26. The method of claim 25,wherein said antibody further comprises a reporter molecule selectedfrom the group consisting of an enzyme and a fluorochrome.
 27. A methodof inhibiting mammalian peroxisomal acetylpolyamine oxidase activitycomprising: a) providing a mammalian peroxisomal acetylpolyamineoxidase, and an inhibitor; and b) contacting said mammalian peroxisomalacetylpolyamine oxidase with said inhibitor under conditions suitablefor reducing amine oxidizing activity of said oxidase.
 28. The method ofclaim 27, wherein said inhibitor is selected from the group consistingof synthalin and N-(3-aminopropyl)-1,10 decanediamine.
 29. The method ofclaim 27, wherein said mammalian peroxisomal acetylpolyamine oxidase islocated in a cell or in an animal.
 30. A method comprising: a) providinga host cell comprising an exogenous nucleic sequence selected from thegroup consisting of the open reading frames of SEQ ID NO:1, SEQ NO:3,SEQ D NO:5 and sequences that hybridize to the complement of the openreading frames of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 under conditionsof low stringency, wherein said isolated nucleic acid sequence encodes apolypeptide having amine oxidizing activity; and b) culturing said hostcell under conditions such that said exogenous nucleic acid sequence isexpressed.